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Metabolic cost of protein synthesis in larvae of the Pacific oyster (Crassostrea gigas) is fixed across genotype, phenotype, and environmental temperature.

Abstract. The energy made available through catabolism of specific biochemical reserves is constant using standard thermodynamic conversion equivalents (e.g., 24.0 J [mg protein.sup.-1]). In contrast, measurements reported for the energy cost of synthesis of specific biochemical constituents are highly variable. In this study, we measured the metabolic cost of protein synthesis and determined whether this cost was influenced by genotype, phenotype, or environment. We focused on larval stages of the Pacific oyster Crassostrea gigas, a species that offers several experimental advantages: availability of genetically pedigreed lines, manipulation of ploidy, and tractability of larval forms for in vivo studies of physiological processes. The cost of protein synthesis was measured in larvae of C. gigas for 1) multiple genotypes, 2) phenotypes with different growth rates, and 3) different environmental temperatures. For all treatments, the cost of protein synthesis was within a narrow range--near the theoretical minimum--with a fixed cost (mean [+ or -] one standard error, n = 21) of 2.1 [+ or -] 0.2 J [(mg protein synthe-sized).sup.-1]. We conclude that there is no genetic variation in the metabolic cost of protein synthesis, thereby simplifying bioenergetic models. Protein synthesis is a major component of larval metabolism in C. gigas, accounting for more than half the metabolic rate in diploid (59%) and triploid larvae (54%). These results provide measurements of metabolic cost of protein synthesis in larvae of C. gigas, an indicator species for impacts of ocean change, and provide a quantitative basis for evaluating the cost of resilience.

Introduction

Many studies have characterized the biochemical composition of early life-history stages of marine invertebrates. For the majority of these species, the organic content of eggs, embryos, and larval stages is dominated by lipid and protein, with carbohydrate being a minor component (< 10%; Holland and Gabbott, 1971; Holland and Spencer, 1973; McClintock and Pearse, 1986; Whyte et al., 1987; Ben-David-Zaslow and Benayahu, 2000; Moran and Manahan, 2004). Calculations of the energy liberated by catabolism of biochemical energy reserves use standard energy equivalents (i.e., lipid: 39.5 J [mg.sup.-1]; protein: 24.0 J [mg.sup.-1]; Gnaiger, 1983; Schmidt-Nielsen, 1997). Data are scant, however, regarding the energetic bases of accumulating biochemical reserves (i.e., cost of biosynthesis). Clearly there are metabolic costs for the biosynthetic processes of development and growth. The study of embryos and larvae of highly fecund marine invertebrates has been foundational to advances in developmental biology, and has revealed complex networks of genes whose spatial and temporal expression patterns regulate rapid rates of protein synthesis and developmental programs (Davidson, 1976, 2010). Since development and protein biosynthesis are so closely linked, supporting the metabolic cost of protein synthesis is a potential constraint on rates of development and growth.

Across animal species, the reported costs of protein synthesis vary widely (two orders of magnitude; Fraser and Rogers, 2007). While the mechanisms underlying this variation in the cost of synthesis measured in whole organisms are unknown, it is clear that no single value can be applied for the anabolic cost of protein synthesis in animals. This uncertainty is in marked contrast to the single value of 24.0 J [mg.sup.-1] that is used for the catabolism of protein. The few studies of the cost of protein synthesis for developmental stages of marine invertebrates show variation between species from different environments (e.g., temperate and polar echinoderms; Marsh et al., 2001; Pace and Manahan, 2006, 2007a, b; Pan et al., 2015a). It remains unknown, however, whether there is genetic variation in the fundamental energetic processes of protein synthesis within a species. If genetically based variation in biosynthesis costs exists within a species, such information would need to be incorporated into bioenergetic models of early development.

The primary goals of this study were to determine the cost of protein synthesis in marine invertebrate larvae, and to test whether this cost was influenced by genotype, phenotype, or environment. We focused on the Pacific oyster Crassostrea gigas (Thunberg, 1793); C. gigas not only allows manipulation of ploidy (Guo et al., 1996), it also offers the distinct advantage of having many genetically pedigreed lines that are the result of decades-long breeding programs (Hedgecock et al., 1995; Hedgecock and Davis, 2007). These lines have been used to study the relationship between genotypic and phenotypic variation in the physiology of larval development (Pace et al., 2006; Meyer and Manahan, 2010; Pan et al., 2015b). Here we measure the cost of protein synthesis in larvae of C. gigas, and test whether this fundamental process varies with genotype, growth phenotype, and environmental temperature. We provide a calculation of energy allocation to protein synthesis during larval development of C. gigas.

Materials and Methods

Source of animals

Diploid ("wild-type") and triploid larvae of Crassostrea gigas were cultured at a commercial shellfish hatchery (Taylor Shellfish Farms, Quilcene, WA). Triploid larvae were produced by mating tetraploid with diploid adults (Guo et al., 1996). Following routine rearing procedures (Breese and Malouf, 1975; Helm and Bourne, 2004), larvae were grown on a mixed algal diet of Isochrysis galbana, Chaetoceros calcitrans, and Tetraselmis suecica. Live larvae (~8-day-old) from five diploid and three triploid cohorts (spawned from different parents and on different dates) were shipped overnight from Taylor Shellfish Farms to the University of Southern California (USC), using standard overnight shipping procedures employed routinely by commercial oyster hatchery operators. Prior to experiments at USC, larvae were held at a density of 10 [ml.sup.-1] in 20-1 culture vessels containing 0.2-[micro]m (pore size)-filtered sea-water at either 20 [degrees]C or 25 [degrees]C (the actual temperature selected depended on experimental requirements).

At the USC Wrigley Marine Science Center (USC-WMSC) on Santa Catalina Island, California, eggs and sperm were removed from the gonads of genetically pedigreed adult broodstock of C. gigas (lines 35 and 51; Hedgecock and Davis, 2007; herein referred to as lines 3 and 5). Broodstock were third-generation inbred ([G.sub.3]) or greater, and the result of ~6 years of breeding and transgenerational culture efforts. In controlled, reciprocal pairwise crosses, hybrid larval families were established (sire x dam: 5x3 and 3 x 5). For larger-scale cultures, two replicate, 200-1 culture vessels per family were set up at an initial concentration of 10 fertilized eggs [ml.sup.-1]. Culture vessels were filled with 0.2-[micro]m (pore size)-filtered seawater and gently aerated to mix the cultures and maintain oxygenation of seawater. At 2 days old, larvae had reached the shelled veliger stage (prodissoconch I) and begun exogenous feeding. Larvae were fed Isochrysis galbana at a concentration of 20,000 cells [ml.sup.-1]. All hybrid families were cultured in seawater maintained at 25 [degrees]C. Culture water was changed every two days. Larvae were collected on appropriate-sized Nitex mesh (Genesee Scientific Corp., San Diego, CA), rinsed of debris using filtered seawater, and returned to clean culture vessels containing freshly filtered seawater and algal food (gradually increased to 50,000 cells [ml.sup.-1] at Day 8). During water changes, local ambient seawater preheated to the required rearing temperature with an inert titanium flow-through heat exchanger was used to refill culture vessels.

Larval shell length and growth

At each sampling period, size-calibrated photomicro-graphs of larvae were taken. The shell length (i.e., distance from the anterior to posterior edge) of 50 or more larvae was measured in micrometers ([micro]m) using ImageJ software (Schneider et al., 2012).

Rates of protein synthesis

Rates of protein synthesis were calculated from time-course assays of the transport and incorporation of a radio-labeled amino acid, using methods developed previously for developmental stages of sea urchin (Pace and Manahan, 2006, 2007a). Due to the differences in composition of the intracellular free amino acid pool between sea urchins and bivalve larvae, the radioisotope [.sup.14]C-glycine was chosen as the tracer for measuring rates of protein synthesis in Crassostrea gigas. For each protein synthesis assay, 10,000 larvae were incubated in 10 ml of filtered seawater (in a 20-ml glass vial), to which was added 74 kilobecquerels (kBq) of [.sup.14]C-glycine (Perkin Elmer, Santa Clara, CA) and a known amount of non-radioactive glycine (Sigma-Aldrich, St. Louis, MO), to bring the final concentration of glycine to 7 [micro]mol [1.sup.-1]. The concentration of glycine used was based on previous studies of the kinetics of amino acid transport by larvae of C. gigas (Manahan, 1989). Aliquots, each containing 1000 larvae of the well-mixed incubation suspension, were removed from the assay vial during a 50-min time-course experiment (n = 6 samples per transport assay). Each aliquot was placed on an 8-[micro]m (pore size) polycar-bonate filter (Nucleopore, Pleasanton, CA) and gently vacuum-filtered to remove seawater that contained radioactivity. Larvae held on the polycarbonate filter were washed three times with filtered seawater to remove any excess radioactivity, placed in a 1.7-ml microcentrifuge tube, immediately frozen on dry ice, and stored at -80 [degrees]C for further processing.

Prior to processing for biochemical analysis, each frozen sample of larvae was resuspended in 500 [micro]l of deionized water (NanoPure model D4754; Barnstead Thermolyne, Dubuque, IA), thawed, held on ice (4 [degrees]C), then sonicated (model VC50 fitted with a microprobe; Sonics & Materials, Inc., Newtown, CT). Aliquots of the homogenate were taken for determination of the intracellular-specific activity of [.sup.14]C-glycine in the free amino acid pool and determination of the total amount of radioactivity incorporated into protein.

Free amino acids were extracted from the homogenate by the addition of ethanol (70% final concentration). The total amount of glycine in each ethanol extract was determined using high-performance liquid chromatography (Pace and Manahan, 2006). The amount of radioactivity in the glycine peak that was collected with an in-line fraction collector (Gilson model FC203B; Gilson, Inc., Middleton, WI) was determined by quench-corrected liquid scintillation counting (Beckman LS6000SC; Beckman Coulter, Brea, CA). The results were used to calculate the rate of change of the intracellular-specific activity of [.sup.14]C-glycine in the free amino acid pool of larvae.

To measure the amount of [.sup.14]C-glycine incorporated into protein, a second aliquot of homogenate was treated with trichloroacetic acid (TCA; 5% final concentration at 4 [degrees]C) to precipitate protein. Precipitated protein was collected on a Whatman GF/C-grade glass microfiber filter (GE Healthcare Bio-Sciences, Pittsburgh, PA), rinsed three times with ice-cold 5% TCA, followed by two rinses with 100% methanol to remove any remaining TCA solution. Filters containing precipitated protein were placed in scintillation vials, scintillation cocktail was added (Ultima Gold, Perkin Elmer), and the amount of radioactivity was counted with appropriate quench correction.

Whole-body total protein for different genotypes representing diploid, triploid (cultured at Taylor Shellfish Farms, Quilcene, WA), and hybrid larvae (USC Wrigley Marine Science Center) was analyzed for amino acid composition at the University of California, Davis, Proteomics Core Facility. Briefly, TCA-precipitated proteins were acid-hydrolyzed with 6 N HC1 for 24 h at 110 [degrees]C, separated by ion-exchange chromatography (Hitachi L-8900; Hitachi, Tokyo, Japan), and quantified spectrophotometrically by post-column derivatization with ninhydrin. From these analyses of the protein amino acids in larvae of C. gigas, two additional measurements were obtained: the mole-percent of glycine (the amino acid used as the tracer of protein synthesis in the experiments described above) and the average molar mass of all amino acids in the proteins. As described in Results, the rate of protein synthesis by larvae of C. gigas was calculated using the 1) change of the specific activity of [.sup.14]C-glycine in the intracellular free amino acid pool, 2) specific activity-corrected rate of incorporation of [.sup.14]C-glycine in larval protein, 3) mole-percent glycine in larval protein, and 4) the average molar mass of all amino acids that comprise larval protein. These calculations yielded rates of protein synthesis expressed as mass of protein synthesized per individual larva per unit of time.

Rates of oxygen consumption

Rates of oxygen consumption were measured using methods previously described (Marsh and Manahan, 1999) and applied to larvae of Crassostrea gigas (Pace et al., 2006). In brief, known numbers of larvae (ranging from 200-400 individuals, depending on size and respiration rate) were placed in oxygen-saturated, 0.2-[micro]m (pore size)-filtered seawater held in gas-tight, micro-biological-oxygen-demand respiration vials. Ten replicate micro-respiration vials were used per assay. The volume of each custom-made glass vial was measured (each vial was ~600 [micro]l). Each micro-respiration vial was held at the desired experimental temperature in a recirculating water bath set at 20 or 25 [degrees]C (depending on the experiment being conducted). Following a known incubation time (range of 3-4 h), the oxygen in each microrespiration vial was measured. This was achieved by injecting a subsample of seawater, removed by a gas-tight syringe (Hamilton Co., Reno, NV), from each micro-respiration vial into a temperature-controlled, oxygen-monitoring unit (MC100 microcell; Strathkelvin, North Lanarkshire, UK). The microcell was fitted with a polarographic oxygen sensor (model 1302; Strathkelvin) connected to an oxygen meter (model 782; Strathkelvin). Oxygen sensor readings were converted to moles of oxygen using standard calibrations. To confirm the estimated number of larvae placed in each micro-respiration vial prior to measurements of oxygen depletion, the individuals in each vial were counted at the end of an assay. Respiration rates were calculated as moles of oxygen consumed per individual larva per unit of time.

Cost of protein synthesis

A major goal of this study was to determine the cost of protein synthesis in larvae of different genotypes. Once the rate of protein synthesis (described above) was obtained, an inhibitor of protein synthesis (emetine) was used. In the presence and absence of emetine, the corresponding changes in rates of protein synthesis and rates of respiration were determined. The difference between the rates of synthesis and respiration in the presence and absence of emetine is a measure of the cost of protein synthesis (see details in Pace and Manahan, 2006, 2007a; Pan et al., 2015a). To test for differences in the cost of protein synthesis as a function of genotype, growth phenotype, and environmental temperature, a series of one-way analysis of variance (ANOVA) tests was used. Prior to ANOVA, data were verified to meet assumptions of normality and equal variance.

Results

Larval growth and size

On separate dates, 8-day-old larvae from separate crosses of 8 different pairs of parents were shipped from the hatchery facility at Taylor Shellfish Farms to USC. Five cohorts of diploid ("wild-type") larvae had mean shell lengths of 141.7 [+ or -] 1.6, 142.4 [+ or -] 0.9, 146.1 [+ or -] 1.3, 151.4 [+ or -] 1.4, or 152.6 [+ or -] 1.1 [micro]m (n = 50-72 larvae measured per cohort). Three cohorts of triploid larvae had mean shell lengths of 146.7 [+ or -] 1.2, 151.1 [+ or -] 1.36, or 151.6 [+ or -] 1.3 [micro]m (n = 49-53 larvae measured per cohort). All error values are reported as one standard error of the mean (SEM) unless otherwise noted. Reciprocal crosses of pedigreed lines of Crassostrea gigas (lines 3 and 5) conducted at USC-WMSC produced families with genetically determined contrasts in growth rate. Growth rates for families 5 x 3 and 3 x 5 were significantly different under the same environmental conditions during the larval rearing period tested (analysis of covariance (ANCOVA), significant interaction between Age and Family indicated a difference in slope of relationship between Age and Shell length for the two families, [F.sub.1,1009] = 54.57, P < 0.0001, n = 1010; Fig. 1). Growth rates were 4.6 [+ or -] 0.16 [[micro]m day.sup.-1] for family 5 x 3 and 3.1 [+ or -] 0.14 [[micro]m day.sup.-1] for reciprocal family 3 x 5. This resulted in a calculated difference of 19.1 jam in shell length of 17-day-old larvae (see Fig. 1 legend for regression equations).

Rates of protein synthesis

The results of the analytical steps required to calculate a rate of protein synthesis are shown in Figure 2 and Table 1. Larvae transport [.sup.14]C-glycine from seawater into the intracellular free amino acid pool. The endogenous pool of non-radiolabeled glycine remains nearly constant during the relatively short period of the transport assay (~50 min); however, the ratio of [.sup.14]C-radiolabeled to non-radiolabeled glycine changes (i.e., the specific activity of glycine in the free amino acid pool changes continuously during the transport assay). Figure 2A shows the relationship between time and the change in specific activity of [.sup.14]C-glycine (see Fig. 2 legend for equation describing the linear rate of change). The measurement of the rate of incorporation of [.sup.14]C-glycine into protein is given in Figure 2B. Since the ratio of moles of non-radiolabeled glycine to [.sup.14]C-glycine is known (Fig. 2A), that correction is applied to the moles of [.sup.14]C-glycine incorporated into protein (Fig. 2B) to quantify the total amount of glycine (both non-radiolabeled and [.sup.14]C-labeled) incorporated into protein from the free amino acid pool.

Calculation of the rate of protein synthesis requires determination of the appropriate mole-percent for the chosen radiolabeled tracer (i.e., glycine in the present study) and the appropriate protein molar mass ([MW.sub.p]) for a given species. Knowing the rate of incorporation of glycine into protein, the ratio of glycine to all amino acids, and the molar mass of whole-body protein of larvae of Crassostrea gigas (Table 1) allows for calculation of the rate of protein synthesis. For wild-type diploid and triploid larvae reared at a commercial shellfish hatchery (Taylor Shellfish Farms), glycine represented 12.0% [+ or -] 0.2% (SEM) of amino acids in protein. For the larvae of reciprocal hybrid families 5 x 3 and 3 x 5, reared at USC (Wrigley Marine Science Center), glycine represented 16.5% [+ or -] 0.3% (SEM) of amino acids in protein. These mole-percent values for glycine were significantly different (ANOVA, [F.sub.1,9] = 241.98, P = 0.001, n = 4-6). When all amino acids were used to calculate [MW.sub.p], the corresponding values were 126.6 [+ or -] 0.2 g [mol.sup.-1] (Taylor Shellfish Farms) and 123.0 [+ or -] 0.3 g [mol.sup.-1] (USC Wrigley Marine Science Center), or a [MW.sub.p] difference of only ~3%, but a difference that was statistically significant (ANOVA, [F.sub.1,9] = 142.69, P = 0.001, n = 4-6).

The specific activity-corrected (Fig. 2A) rate of incorporation of glycine into protein (Fig. 2B) can be converted to the mass of protein synthesized with time, following correction for mole-percent glycine and [MW.sub.p] (Table 1). The slope of the regression relationship for cumulative protein synthesized with time is the absolute rate of protein synthesis (Fig. 2C). For example, between 8 and 16 min of the assay shown in Figure 2B a total of 32.4 mBq of [.sup.14]C-glycine was incorporated into larval protein (i.e., 54.9 mBq [larva.sup.-1] at 16 min minus 22.5 mBq [larva.sup.-1] at 8 min; Fig. 2B). The mean specific activity for the same interval was 36.2 mBq [pmol.sup.-1] (i.e., average of 24.3 mBq [pmol.sup.-1] at 8 min and 48.1 mBq [pmol.sup.-1] at 16 min; Fig. 2A). Correcting the amount of [.sup.14]C-glycine incorporated for the change in specific activity over this time interval (i.e., 32.4 mBq [larva.sup.-1] divided by 36.2 mBq [pmol.sup.-1]) yielded a total incorporation of 0.9 pmol glycine [larva.sup.-1] during the 8-16-min interval of the assay. This calculation was repeated for each subsample in the time-course experiment to correct the amount of [.sup.14]C-glycine incorporated to total moles of glycine. The moles of glycine were, in turn, converted to the amount of protein synthesized using the measured values for the mole-percent glycine in larval protein and the [MW.sub.p] of protein (Table 1). This conversion was as follows: 0.9 pmol glycine divided by 0.12 (mole-percent glycine, 12.0%) in protein gave 7.5 pmol of all amino acids incorporated into protein. Multiplying this value by the molar mass of protein ([MW.sub.p], 126.6 g [mol.sup.-1], Table 1) 0.9 ng protein synthesized between 8 and 16 min. The slope of the cumulative amount of protein synthesized for each interval gave a synthesis rate of 6.2 [+ or -] 0.4 ng protein [larva.sup.-1] [1.sup.-1] (error term is standard error (SE) of the regression slope; Fig. 2C).

The cost of protein synthesis

Inhibition of protein synthesis. The efficacy of the protein synthesis inhibitor, emetine, in larvae of Crassostrea gigas was assessed over a range of concentrations (0.5-200 [micro]mol [1.sup.-1]). Compared to the protein synthesis rates in controls (i.e., larvae not treated with emetine), synthesis declined steeply with increased concentrations of emetine. Near-maximum inhibition occurred at 25 [micro]mol [l.sup.-1], with no significant decrease measurable when the concentration of emetine was further increased to 100 [micro]mol [1.sup.-1] (ANOVA of regression of slope: P = 0.772, [R.sup.2] = 0.05, df = 3; Fig. 3A). A concomitant reduction in respiration rate also occurred in larvae treated with emetine at 25 [micro]mol [l.sup.-1] (Fig. 3B). Higher concentrations of emetine, however, decreased the respiration rate (significant negative slope of regression, ANOVA of regression of slope: P < 0.001, [R.sup.2] = 0.29, df = 44; Fig. 3B), indicating that emetine had non-specific effects on respiration at higher concentrations. For the experiments reported here, an emetine concentration of 25 [micro]mol [l.sup.-1] was used, which effectively inhibited protein synthesis while minimizing the non-specific effects on respiration.

Measurement of the cost of protein synthesis. The cost of protein synthesis was determined from an analysis of emetine-induced decreases in rates of protein synthesis and respiration conducted in parallel on aliquots of larvae from the same culture (Fig. 4). In this representative experiment (from a total of 21 sets of assays; Fig. 5), emetine reduced the rate of protein synthesis from (mean [+ or -] SE of the regression slope) 2.9 [+ or -] 0.3 ng protein [larva.sup.-1] [1.sup.-1] in control larvae to 0.8 [+ or -] 0.1 ng protein [larva.sup.-1] [1.sup.-1] in the presence of emetine--a decrease of 2.1 ng protein [larva.sup.-1] h. The corresponding decrease in respiration rate was from 29.6 [+ or -] 1.7 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1] in controls to 21.8 [+ or -] 1.4 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1] in the presence of emetine--a reduction of 7.8 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1] (Fig. 4, inset). Converting oxygen to energy equivalents using a value of 484 kJ [(mol [O.sub.2]).sup.-1] (i.e., the average oxyenthalpic equivalent for lipid and protein, which are the major biochemical reserves in larvae of C. gigas), a respiration rate of 7.8 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1] equates to 3.8 [micro]J. From these data, a cost of protein synthesis can be calculated. A difference of 2.1 ng protein-synthesized [larva.sup.-1] [1.sup.-1] with a concurrent difference in respiration of 3.8 [micro]J results in a calculated cost of protein synthesis of 1.8 J [(mg protein synthesized).sup.-1]. A total of 21 such assays were conducted on larval families of C. gigas of different genotypes, level of ploidy, and temperatures (20 [degrees]C and 25 [degrees]C) (Fig. 5). In these 21 separate sets of experimental trials (e.g., Figs. 2, 4), the measured cost of protein synthesis fell within a narrow range of 1.0-3.7 J [(mg protein synthesized).sup.-1]--within estimates of the theoretical minimum, which span 1-5 J [(mg protein synthesized)".sup.1] (see Discussion).

A series of statistical analyses were performed to test whether the cost of protein synthesis varied as a function of 1) genotype, 2) growth phenotype, or 3) environmental temperature. Four genotypes were tested (diploid, triploid, and hybrid families 5 x 3 and 3 x 5). Pretesting the data for assumptions of ANOVA (Shapiro-Wilk normality test, P = 0.340; equal variance, P < 0.050), a non-parametric Kruskal-Wallis One-Way ANOVA on rank was used. There was no effect of genotype on the cost of protein synthesis (Kruskal-Wallis H statistic = 5.323, df = 3, P = 0.150). Families 5 x 3 and 3 x 5, which had contrasting growth phenotypes, were tested for differences in the cost of protein synthesis using one-way ANOVA (Shapiro-Wilk normality test, P = 0.277; equal variance, P = 0.066). There was no effect of growth phenotype on the cost of protein synthesis (ANOVA, [F.sub.1, 7] = 0.900, P = 0.379). There was also no effect of temperature on the cost of protein synthesis (Sha-piro-Wilk normality test, P = 0.084; equal variance, P = 0.083; ANOVA, [F.sub.1,20] = 0.247, P = 0.625). In conclusion, there are no significant differences in the cost of protein synthesis for genotype, growth phenotype, or environmental temperature (Fig. 5). In addition, in comparing all 21 groups representing a combination of genotype, growth phenotype, and temperature, there is no difference in the cost of protein synthesis (one-way ANOVA, [F.sub.5, 20] = 2.395, P = 0.087). We conclude that larvae of C. gigas have a fixed cost of protein synthesis of 2.1 [+ or -] 0.2 J [(mg protein synthesized).sup.-1].

Allocation of metabolic energy to support protein synthesis. The rates of protein synthesis and respiration for two agecohorts of larvae (mean shell lengths of 142.4 [micro]m and 151.6 [micro]m) were used to calculate the energy allocated to synthesizing protein at a cost of 2.1 J [(mg protein synthesized)".sup.1]. The proportion of respiration accounted for by rates of protein synthesis in diploid and triploid larvae of C. gigas was 59% and 54%, respectively (Table 2).

Discussion

Maintaining homeostasis under routine conditions or in response to environmental perturbations necessitates the regulated distribution of available energy (ATP) to meet the demands of multiple physiological processes. Measuring ATP required for specific biochemical processes underlying the cost of living is an important theme in the study of physiological adaptation (Schmidt-Nielsen, 1997; Hochachka and Somero, 2002). Ion pump activity and protein synthesis generally dominate ATP use (Siems et al., 1984, 1992; Rolfe and Brown, 1997; Wieser and Krumschnabel, 2001; Hochachka and Somero, 2002; Pan et al., 2015a). Despite an extensive literature on rates and mechanisms of protein synthesis (reviewed by Fraser and Rogers, 2007), the costs of synthesis in developmental stages of marine animals have been quantified for only a few species of invertebrates (e.g., sea urchins, Marsh et al., 2001; Pace and Manahan, 2006, 2007a, b; Pan et al., 2015a) and fish (Houlihan et al., 1995). Further, considerable genetically determined variation in biochemical and physiological rates within a species (Pace et al., 2006; Pan et al., 2015b) occurs even under controlled environmental conditions. Here we tested whether there was genetic variation in the metabolic cost of protein synthesis, a fundamental biosynthetic process.

Metabolic cost of protein synthesis

Across 21 separate trials (each including multiple timecourse protein synthesis assays and independent sets of respiration measurements; Figs. 2, 4) with larvae of Crassostrea gigas representing different genotypes, growth phenotypes, and environmental temperatures, the cost of protein synthesis was consistently near the theoretical minimum. Individual values fell within a narrow range of between 1.0 and 3.7 J [(mg protein synthesized).sup.-1], and averaged 2.1 [+ or -] 0.2 J [(mg protein synthesized).sup.-1] across all treatments. This narrow range contrasts with the empirical values that span more than two orders of magnitude. On a theoretical basis, the minimum cost of protein synthesis is estimated at between ~1 and ~5.0 J [mg.sup.-1] (Buttery and Boorman, 1976; Webster, 1981; Pace and Manahan, 2007a), with calculations of a specific cost varying depending on 1) the value used for energy yield (J) per mole of ATP (e.g., 30.5 kJ [mol.sup.-1], Lehninger, 1975; 46-54 kJ [mol.sup.-1], Alberts et al., 1983; 75.5 kJ [mol.sup.-1], Aoyagi et al., 1988); 2) the values used for amino acid composition of total protein across different species (e.g., g [mol.sup.-1] for C. gigas; Table 1); and 3) the use of appropriate oxyenthalpic equivalents that account for the relative proportions of carbohydrate, lipid, and protein used by a species or developmental stage to fuel metabolism (Gnaiger, 1983). In the current study, across all trials the cost of synthesis did not differ significantly, and averaged 2.1 [+ or -] 0.2 J [(mg protein synthesized).sup.-1] (Fig. 5). This fixed cost of protein synthesis in larvae of C. gigas agreed with earlier findings of fixed costs during echinoderm development and growth (Pace and Manahan, 2006, 2007a, b), and is essentially the same as the value recently reported for a temperate species of sea urchin, Strongylocentrotus purpuratus: 2.4 [+ or -] 0.2 J [(mg protein synthesized).sup.-1]--a cost that is also fixed for multiple developmental stages of sea urchin as well as under simulated conditions of ocean acidification (Pan et al., 2015a).

A wide range of values is reported in the literature for the cost of protein synthesis in animals measured in vivo. These values span more than two orders of magnitude (Fraser and Rogers, 2007), from 0.45 J [(mg protein synthesized).sup.-1] for Antarctic sea urchin, near the theoretical minimum (Marsh et al., 2001; Pace and Manahan, 2007a), to 81.7 J [mg.sup.-1] for the Antarctic isopod (based on 5.2 ATP mol [O.sub.2.sup.-1] and 480 kJ mol [O.sub.2.sup.-1] for 885 mmol ATP [g.sup.-1], as reported by Whiteley et al., 1996). Examples of adult marine animals include mussels (11.4 J [mg.sup.-1], Hawkins et al., 1989) and crabs (18.8 J [mg.sup.-1], based on 39.1 mmol [O.sub.2] [g.sup.-1] reported by Houlihan et al., 1990, using a conversion of 480 kJ mol [O.sub.2.sup.-1]). In general., these values are in the range reported for terrestrial animals (e.g., chickens at 5.4 J [mg.sup.-1] in Aoyagi et al., 1988 and 13.0 J [mg.sup.-1] in Muramatsu and Okumura, 1985), including mammals (11.5-34.9 J [mg.sup.-1], Reeds et al., 1985; Coyer et al., 1987; Fuller et al., 1987). The bases for the large variations in cost of protein synthesis are unclear, particularly for those values that extend more than an order of magnitude above the theoretical minimum. While true biological variation may account for some of this range, it is likely that technical challenges in measuring biochemical and physiological rates in vivo are a major contributor to the wide range of values reported in the literature for the cost of protein synthesis. It is worth noting that when using in vitro cell-free systems, values of 5.6 and 4.3 ATP per peptide bond have been reported for tissues of temperate and Antarctic scallops, respectively (Storch and Portner, 2003). Using the equation from Aoyagi et al. (1988), as cited by Storch and Portner (2003), these ATP cost values equate to 4.0 and 3.1 J [(mg protein synthesized).sup.-1], which are within the range of theoretical minimum cost values. This in vitro approach presumably reduces or eliminates potential contributions from physiological costs other than those associated with protein synthesis, where the latter may inflate the measured cost of synthesis.

The literature supports much greater concordance among theoretical estimates, results from in vitro assays (Storch and Portner, 2003), and costs of protein synthesis measured in vivo for the small embryos and larvae of marine and freshwater invertebrates and fish (Houlihan et al., 1993; Conceicao et al., 1997; Marsh et al., 2001; Pace and Manahan, 2006, 2007a, b; Pan et al., 2015a, but see Houlihan et al., 1995 for a higher cost of synthesis in larvae of herring). We speculate that this concurrence of theoretical and measured values is due to advantages inherent in working with small developmental stages, which facilitate measurements of rates and costs in vivo. Developing marine invertebrates in particular have 1) rapid uptake from the experimental medium of the radioactively labeled substrates used to measure biosynthesis (Stephens and Schinske, 1961; Manahan, 1990); 2) less morphological complexity than larger animals, which facilitates uniform distribution of tracer molecules (tRNA loading; Regier and Kafatos, 1977); 3) tractability to perfuse cells with metabolic inhibitors; and 4) sufficiently small size for whole-organism level in vivo measurements.

Physiological studies of larvae of Crassostrea gigas

The Pacific oyster is one of the most aquacultured species on the planet, and is found in coastal ocean waters around six of the seven continents (Mann, 1979; Food and Agriculture Organization of the United Nations, 2012). Interest in understanding the development, growth, and survival of larvae of Crassostrea gigas has resulted in many studies of the biochemistry and physiology of this species. In our study, the primary data values (Figs. 2-4; Table 1) for respiration and protein synthesis used to calculate the cost of synthesis in larvae of C. gigas were consistent with other independent reports. Metabolic rates of oyster larvae have been measured with indirect calorimetry (oxygen consumption, Gerdes, 1983; Hoegh-Guldberg and Manahan, 1995; Pace et al., 2006) and direct calorimetry (rates of heat dissipation, Widdows et al., 1989; Hand, 1999). Biochemically, changes in the relative proportions of carbohydrate, lipid, and protein are well characterized in larvae of C. gigas, with carbohydrate being a small component of energy reserves (~5%, Moran and Manahan, 2004). Protein content increases from ~60% of measured biochemical composition in 1-day-old larvae to ~80% from Day 6 onward (His and Maurer, 1988; Moran and Manahan, 2004). The lipid proportion decreases from ~19% to less than 10% just before metamorphosis (His and Maurer, 1988; Laing and Earl, 1998). Bioenergetic models have been developed for larval stages of C. gigas (Bochenek et al., 2001). There is also genetically determined variation in growth rate and other biochemical and physiological processes in larvae of C. gigas (Pace et al., 2006; Pan et al., 2015b).

Respiration. Our measured rates of oxygen consumption that were used to calculate the cost of protein synthesis for larvae of C. gigas are consistent with previous results for respiration rates for this species (Pace et al., 2006). For example, a verification of respiration rates for the diploid larvae used in the current study (shell length of 142.4 [+ or -] 0.9 [micro]m; Table 2) can be obtained based on a size-specific comparison with the respiration rates measured at 23 [degrees]C by Pace et al. (2006). Applying a standard [Q.sub.10] value (temperature coefficient) of 2.0 to adjust respiration rates at 23 [degrees]C to rates at 25 [degrees]C (the measurement temperature in the current study) yields a rate of 40.6 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1]-a value that compares well with the measured rate 45.4 [+ or -] 2.0 pmol [O.sub.2] [larva.sup.-1] [1.sup.-1] (Table 2).

Synthesis rates. We know of no other measurements for rates of protein synthesis in larvae of C. gigas; however, a calculation of fractional rates of synthesis can be obtained for comparison to other developmental stages of marine invertebrates (e.g., echinoderms). A diploid larva of C. gigas with shell length 142.4 [+ or -] 0.9 [micro]m (Table 2) and protein synthesis rate of 6.2 [+ or -] 0.4 ng [1.sup.-1] (Fig. 2C; Table 2) contains an estimated 101 ng protein (regression equation of shell length and protein content from Pace et al., 2006). The ratio of the protein content per larva (101 ng) to the synthesis rate (6.2 ng h ) is the fractional synthesis rate, and is 6.1% [1.sup.-1]. This value accords reasonably well with rates measured for feeding larval stages of marine invertebrates that have high rates of cell division and fast growth (Pace and Manahan, 2006, 2007b; Fraser and Rogers, 2007), although it should be noted that the rate for C. gigas was measured at a higher temperature (25 [degrees]C).

Application of protein synthesis inhibitors. In our study, the cost of synthesis was determined from the concurrent reduction in metabolic rate and protein synthesis in the presence of a specific inhibitor of protein synthesis (Figs. 3, 4). Inhibition of protein synthesis was achieved using emetine, a rapid and irreversible inhibitor of protein synthesis. Emetine interacts with the 40S ribosomal subunit and blocks translation by stabilizing the ribosomal complex, halting the aminoacyl-tRNA transfer reaction and preventing the peptide chain and mRNA from leaving the ribosomal complex (Gupta and Siminovitch, 1977). Emetine has been used to inhibit protein synthesis in other marine invertebrates (Epel, 1972; Fenteany and Morse, 1993; Pace and Manahan, 2006, 2007a; Villareal et al., 2007; Pan et al., 2015a). An important assumption is that inhibition is specific to protein synthesis, at least within the timescale of concurrent measurements of protein synthesis and metabolic rate. We tested for inhibition of protein synthesis and respiration rates in larvae of C. gigas over a range of emetine concentrations (0.5-200 [micro]mol [1.sup.-1]; Fig. 3). Protein synthesis declined significantly compared with controls (when no emetine was present) at the lower concentrations tested, but synthesis rates did not further decline between 25 and 100 [micro]mol [1.sup.-1] emetine (Fig. 3A). Respiration was also significantly inhibited at the low concentrations of emetine (Fig. 3B). In contrast to protein synthesis, however, a continued and significant decline in respiration occurred between 50 and 200 [micro]mol [1.sup.-1] emetine. Increased inhibition of respiration in the absence of further inhibition of protein synthesis suggests that non-specific effects may occur at higher concentrations of emetine. This result illustrates the importance of testing and carefully selecting the concentration of protein synthesis inhibitors used, such that they elicit the desired, process-specific response.

Choice of radioactively labeled amino acid

To calculate rates of protein synthesis, incorporation of [.sup.14]C-glycine in larval protein was corrected for changes in specific activity of the intracellular free amino acid pool (Fig. 2A). The resulting incorporation of moles of glycine (Fig. 2B) was converted to mass of total protein synthesized (Fig. 2C), based on the mole-percent glycine and the amino acid composition of total protein (Table 1). It is noteworthy that there is a significant difference in the mole-percent of glycine in larval protein, and in the mole-percent calculation of amino acids constituting protein mass, for larvae reared in different hatcheries (USC Wrigley Marine Science Center, CA; Taylor Shellfish Farms, WA). The amino acid composition of whole-body protein reported by Brown (1991) for larvae of Crassostrea gigas was similar (at 124.0 g [mol.sup.-1]) to the values that we report in Table 1. Further analyses of the protein amino acid composition of developmental stages from different cohorts reared under multiple environmental conditions would be valuable.

Allocation of metabolic energy to support protein synthesis

Our results provide measurements of the rate and metabolic cost of protein synthesis in larvae of Crassostrea gigas--a species that is the basis of one of the largest global marine aquaculture industries (Food and Agriculture Organization of the United Nations, 2012), and for which the developmental stages have emerged as an indicator of the impacts of ocean acidification (Barton et al., 2012, 2015; Waldbusser et al., 2015). We have established that the cost of synthesizing protein is fixed at 2.1 J [mg.sup.-1] (Fig. 5) for 1) different genotypes (diploid, triploid, and reciprocal hybrid families), 2) contrasting phenotypes under controlled environmental conditions (different growth rates), and 3) a fundamental environmental regulator of physiological rate (different temperatures). Our results indicate that protein synthesis can account for more than half of total metabolic energy. It is noteworthy that for wild-type diploid larvae representative of natural populations, or triploid larvae that have been manipulated to facilitate increased yield in commercial aquaculture, the cost of protein synthesis is the same (Fig. 5), as is the proportion of metabolic energy allocated to protein synthesis (59% and 54% for similar-sized diploid and triploid larvae, respectively; Table 2). While the protein synthesis cost is fixed, it remains unknown to which degree the percent allocation of total metabolic energy to protein synthesis can vary in response to environmental conditions, or whether allocation varies among genotypes. Such variation has major implications for understanding the mechanistic bases of growth and survival., and the energy limits of resilience to environmental stress. Having now determined the cost of protein synthesis in this species, we have made a requisite step towards testing these hypotheses.

Substantial variations in growth rate of marine invertebrate larvae are prevalent, even within a single age-cohort of larvae reared under the same controlled environmental conditions (Strathmann, 1987). A large portion of phenotypic variation in larvae can be attributed to genetic differences; the use of pedigreed lines of the Pacific oyster (Hedgecock et al., 1995; Hedgecock and Davis, 2007) has facilitated exploration of the genetic and physiological bases of variation of phenotypic traits in larvae (Pace et al., 2006; Curole et al., 2010; Meyer and Manahan, 2010; Pan et al., 2015b). Previously, a metabolic model was used to illustrate how variation in protein depositional efficiency could account for a major portion of the energy required for differential growth among larval families of Crassostrea gigas (Pace et al., 2006). An implicit assumption of that model is that the cost of protein synthesis does not differ among the families (i.e., among genotypes). Here we have validated that assumption and, furthermore, we have provided the actual cost of protein synthesis for larvae of C. gigas. Having shown that the cost of synthesis is fixed, we have also constrained the possible physiological and biochemical mechanisms of genetically determined variation in growth rates (Bochenek et al., 2001; Pace et al., 2006).

The biological responses of marine organisms to future scenarios of global environmental change are of great interest. Increasing temperatures and changes in seawater carbonate chemistry (ocean acidification) both present major concerns for the future of marine animal populations in the wild and those being systematically produced in marine aquaculture (Parmesan, 2006; Doney et al., 2009; Food and Agriculture Organization of the United Nations, 2009; Hoegh-Guldberg and Bruno, 2010; Somero, 2010, 2012; Sunday et al., 2014). Changes observed at the level of the whole organism (e.g., reduced growth, survival., reproduction) in response to environmental stressors are frequently presumed to be associated with increased energy demands (Sokolova et al., 2012; Stumpp et al., 2012; Pespeni et al., 2013; Ramajo et al., 2016). However, quantitative measures of these energy costs are difficult to make, and are rarely undertaken.

Nonetheless, this approach offers the advantage of allowing a quantitative comparison of sublethal biological responses under a range of future environmental scenarios. For example, in growing sea urchin larvae allocation of energy to protein synthesis and ion transport increased from an average of 55% under present-day conditions to 84% under a near-future ocean acidification scenario, with the majority of this increase accounted for by changes in the rate of protein synthesis but not respiration (Pan et al., 2015a). Defining the complex trade-offs in allocation of cellular energy between the routine cost of living and the additional cost imposed on organisms by having to respond to compounding environmental stressors will require an understanding of both the genetic and physiological bases of adaptation (Somero, 2010; Applebaum et al., 2014). Such approaches will help establish the key mechanisms and concomitant energy costs of resilience to environmental change.

Acknowledgments

Dr. Dennis Hedgecock assisted with genotyping of adult broodstock used for crosses of pedigreed lines, and also provided helpful advice. We thank Dr. Jonathan Davis and Taylor Shellfish Farms for their support and for supplying some of the biological materials used in these experiments. This work was supported by a grant from the U.S. National Science Foundation (Emerging Frontiers no. 121220587).

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JIMMY W. LEE([dagger]), SCOTT L. APPLEBAUM([dagger]), AND DONAL T. MANAHAN (*)

Department of Biological Sciences, University of Southern California, Los Angeles, California 90089

Received 2 February 2016; accepted 11 April 2016.

(*) To whom correspondence should be addressed. E-mail: manahan@usc.edu

([dagger]) Authors JWL and SLA contributed equally to this work.

Table 1
Mole-percent amino acid composition and average protein molar mass
([MW.sup.p]) for larvae of the Pacific oyster Crassostrea gigas reared
in hatcheries at Taylor Shellfish Farms (Quilcene, WA) or at the USC
Wrigley Marine Science Center (Santa Catalina Island, CA)

                            Taylor                   Wrigley
Amino acid            (% [+ or -] SEM. n = 6)  (% [+ or -] SEM, n = 4)

Alanine                 7.8 [+ or -] 0.1         8.0 [+ or -] 0.2
Arginine                5.6 [+ or -] 0.1         5.1 [+ or -] 0.0
Aspartate/asparagine   10.5 [+ or -] 0.1        10.0 [+ or -] 0.0
Glutamate/glutamine    11.0 [+ or -] 0.1        10.0 [+ or -] 0.2
Glycine                12.0 [+ or -] 0.2        16.5 [+ or -] 0.3
Histidine               1.9 [+ or -] 0.0         1.6 [+ or -] 0.1
Isoleucine              5.1 [+ or -] 0.1         4.7 [+ or -] 0.1
Leucine                 8.1 [+ or -] 0.1         7.6 [+ or -] 0.1
Lysine                  8.2 [+ or -] 0.1         6.7 [+ or -] 0.3
Methionine              0.9 [+ or -] 0.1         0.8 [+ or -] 0.0
Phenylalanine           4.0 [+ or -] 0.0         3.8 [+ or -] 0.1
Proline                 3.2 [+ or -] 1.0         4.6 [+ or -] 0.1
Serine                  6.1 [+ or -] 0.1         6.3 [+ or -] 0.2
Threonine               5.6 [+ or -] 0.1         5.3 [+ or -] 0.1
Tyrosine                3.6 [+ or -] 0.2         3.4 [+ or -] 0.0
Valine                  6.3 [+ or -] 0.2         5.7 [+ or -] 0.1
MWp(g [mol.sup.-1])   126.6 [+ or -] 0.2       123.0 [+ or -] 0.3

The mole-percent glycine and the MWp (table entries shown in bold)
were used to calculate rates of protein synthesis. To calculate
[MW.sup.p], mole-percent values were multiplied by the respective
molecular weight of each amino acid and these values were summed. A
total of 18 of 20 proteinogenic amino acids are accounted for in this
analysis, as cysteine and tryptophan are not detected by this
analytical scheme. However, these molecules constitute one mole-percent
or less of total protein in larvae of Crassostrea gigas (Brown, 1991),
and their inclusion in the calculations would affect both glycine
mole-percent and MWp by 0.5 units or less. Asparagine and glutamine
form aspartate and glutamate, respectively, during the acid hydrolysis
analysis step.

Table 2
Proportion of metabolic energy allocated to protein synthesis in
wild-type diploid and triploid larvae of Crassostrea gigas

                                                           Diploid

Shell length ([micro]m)                            142.4 [+ or -] 0.9
Respiration (pmol [larva.sup.-1] [1.sup.-1])        45.4 [+ or -] 2.0
Protein synthesis (ng larva-1 [1.sup.-1])            6.2 [+ or -] 0.4
Cost of protein synthesis (J mg [protein.sup.-1])    2.1 [+ or -] 0.2
Metabolic rate ([micro]J [larva.sup.-1]
[day.sup.-1])                                      527
Total cost to synthesize protein                   310
([micro]J [larva.sup.-1] [day.sup.-1])
Energy allocation to protein synthesis (%)          59

                                                          Triploid

Shell length ([micro]m)                            151.6 [+ or -] 1.3
Respiration (pmol [larva.sup.-1] [1.sup.-1])        43.3 [+ or -] 2.3
Protein synthesis (ng larva-1 [1.sup.-1])            5.4 [+ or -] 0.4
Cost of protein synthesis (J mg [protein.sup.-1])    2.1 [+ or -] 0.2
Metabolic rate ([micro]J [larva.sup.-1]
[day.sup.-1])                                      503
Total cost to synthesize protein                   271
([micro]J [larva.sup.-1] [day.sup.-1])
Energy allocation to protein synthesis (%)          54

Metabolic rates were calculated from measured respiration rates using
484 kJ (mol O2)-1, the average oxyenthalpic equivalent for lipid and
protein (the major biochemical reserves in larvae of C. gigas).
Experimentally measured values are reported as means [+ or -] 1 SEM.
All other values were calculated from measured values. Values in the
table are from experiments conducted with two age-cohorts of larvae
(diploid and triploid) at 25 [degrees]C.


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Author:Lee, Jimmy W.; Applebaum, Scott L.; T. Manahan, Donal
Publication:The Biological Bulletin
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Date:Jun 1, 2016
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