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Cost of protein synthesis and energy allocation during development of Antarctic sea urchin embryos and larvae.


The relationship between temperature and metabolism of animals is an important theme in environmental physiology (Portner and Playle, 1998; Hochachka and Somero, 2002; Portner, 2006). For metabolic adaptations of animals living in cold environments, the interpretation of measured respiration rates has been debated for many decades, starting with the pioneering work of Krogh (1914) and the concept of metabolic cold adaptation (Scholander et al., 1953; Wohlschlag, 1960; Holeton, 1974; Clarke, 1991; Peck and Conway, 2000). In polar oceans, research has revealed unique evolutionary mechanisms of biological adaptation to extreme cold. For instance, Antarctic fish have evolved antifreeze glycoproteins to prevent freezing at low temperatures (DeVries, 1971; Chen et al., 1997). Other notable physiological strategies in Antarctic animals include cold adaptation of microtubule motors (Detrich et al., 1992), low enzyme-specific activity of ion pumps (Leong and Manahan, 1999), loss of inducible heat-shock response (Hofmann et al., 2000), loss of respiratory proteins (Moylan and Sidell, 2000; di Prisco et al., 2002), high enzyme activity of lipase (Sidell and Hazel, 2002), and partial temperature compensation of enzymes involved with ATP-generating pathways (Kawall et al., 2002).

Larval forms are dominant in the life-history strategies of most marine animals (Thorson, 1950; Cowen et al., 2006). During development and growth of larvae, major changes occur rapidly in size and form, and in biochemical and physiological rates (Zeuthen, 1953; Davidson, 1986). Such processes have been characterized in many species of larvae from temperate environments (Widdows, 1991; Hoegh-Guldberg and Manahan, 1995). Additionally, for some species of marine invertebrates the major biochemical mechanisms determining metabolic costs during development and growth have been quantified (Leong and Manahan, 1997; Pace and Manahan, 2006). In contrast, far less is known about developmental physiology in cold environments. Given that a very large percentage of the living biosphere (including deep-sea environments) is cold (< 4 [degrees]C), a better understanding of the metabolic costs of development, growth, and the "cost of living" in cold environments is warranted.

Details of the biochemical bases for regulation of metabolic rates in animals have, for the most part, remained elusive (Kleiber, 1961; Heusner, 1991). Protein turnover--the synthesis and degradation of proteins--is certainly one of the major components that sets metabolic rate (Waterlow, 1984; Hawkins, 1991; Houlihan, 1991). Protein synthesis can account for a wide and significant range of metabolism in several marine organisms (cod: 24%-42%, Houlihan et al., 1988; octopus: 35%-51%, Wells et al., 1983; mussels: 16%, Hawkins, 1985). Such synthesis costs can be variable, as in fish where the cost of protein synthesis varies as a function of the rate of synthesis--that is, high rates result in relatively lower costs (Pannevis and Houlihan, 1992; Smith and Houlihan, 1995). In developmental stages of temperature sea urchins, costs of protein synthesis are fixed and can account for 40%-54% of metabolism during embryogenesis (Pace and Manahan, 2006).

Rates and costs of protein synthesis have been measured in adult stages of Antarctic marine invertebrates. Rates of synthesis in an Antarctic isopod were relatively low compared to those in a temperate isopod, with cost of synthesis being 4 times higher in the Antarctic species (Whiteley et al., 1996). These authors suggested that the low rates of synthesis measured in the Antarctic isopod were a result of the extremely high cost of synthesis in the cold. Fraser et al. (2002) reported rates of synthesis in an Antarctic limpet to be similar to rates in temperate molluscs. When converted to energy equivalents on the basis of use of a theoretical minimum cost of protein synthesis, the authors estimated that protein synthesis accounted for 34%-40% of oxygen consumption, a range that supports the general conclusion of high metabolic costs of protein synthesis. Storch and Portner (2003) reported similar costs of protein synthesis for translationally active cell-free homogenates prepared from gills of adult Antarctic scallops when compared to homogenates from European scallops. In early life-history stages of Antarctic organisms, Shilling and Manahan (1994) found that the low respiration rates of embryos and larvae of echinoderms allowed for survival for months to years without food (cf. days to weeks for comparable temperate species). Surprisingly, this long potential life span of developmental stages in the cold is not related to low respiration rates set by low biosynthetic rates: rates of biosynthesis (RNA and protein) are high in developing Antarctic sea urchins (protein turnover at ~2% [h.sup.-1]: Marsh et al., 2001). This rate of turnover at a cold temperature of -1.5 [degrees]C is essentially the same as the rate measured in well-characterized species of sea urchins developing at much higher temperatures of 15-25 [degrees]C (Berg and Mertes, 1970; Fry and Gross, 1970; Goustin and Wilt, 1981).

In this study we measured rates and costs of protein synthesis in a range of embryonic and larval stages of the Antarctic sea urchin Sterechinus neumayeri (Meissner, 1900). We show that costs of protein synthesis are fixed and uniquely low in this species. Combined with previous studies from our laboratory on the metabolic importance of the sodium pump during development of this Antarctic sea urchin, we now provide an analysis of the "cost of living" during development in the cold. Up to 87% of the metabolic rate can be accounted for by the combined energy costs of protein synthesis and the sodium pump for this organism living in an extreme-cold environment.

Materials and Methods

Animal culturing

Adult specimens of Sterechinus neumayeri were collected from McMurdo Sound, Antarctica, by scuba divers. Release of gametes was induced using standard methods to spawn sea urchins (injection of 0.5 mol [l.sup.-1] KCl). Only cultures that were initiated from eggs showing elevation of fertilization envelopes in over 90% of the eggs within 5-10 min of sperm addition were used in this study. Fertilized eggs were placed in 20-liter culture vessels at concentrations of 5-10 [ml.sup.-1]. All seawater used in culturing and experiments was collected from McMurdo Sound, sterile-filtered (pore size 0.2 [micro]m, Nucleopore), and maintained at ambient temperature in the aquarium at McMurdo Station. Motorized rotating paddles were used to keep embryos and larvae gently suspended. Culture water was changed every 3 days by gently collecting larvae on mesh screens submerged in ambient-temperature seawater.

Amino acid transport and rate of protein synthesis

Absolute rates of protein synthesis were measured in embryos and larvae of S. neumayeri by using tracer amounts of radioactive amino acids. Experiments using small additions of radioactive substrates have been used extensively for several decades to quantify many biochemical rate processes (e.g., DNA and RNA synthesis, protein synthesis) in developing stages of echinoderms (Epel, 1967; Fry and Gross, 1970; Regier and Kafatos, 1977; Wagenaar, 1983; Yamada, 1998; Pace and Manahan, 2006; also reviewed in Davidson, 1986). We used a "tracer-dose" method for our studies of protein synthesis because sea urchin embryos have large pools of free amino acids, as is common among marine invertebrates (Yancey et al., 1982). Other methods for measuring protein synthesis rates, such as the "flooding-dose" method (Garlick et al., 1980), are not practical for use in organisms with high amounts of intracellular free amino acids (Berg, 1965, 1968; discussed in Davidson, 1986, appendix III). Flooding dose methods have been used in some organisms to reduce the possibility of intracellular compartmentalization leading to inaccuracy when using isotopes to measure protein synthesis rates. However, Regier and Kafatos (1977) determined that the specific activity of free amino acid and aminoacyl-tRNA pools in sea urchins embryos were similar when tracer amounts of radioactive amino acids were used to measure synthesis rates (i.e., there was no measurable compartmentalization). A further rationale for using tracer dose amounts of radiolabeled amino acids when working with embryos and larvae is that cellular perturbation by added substrates is minimized (see Davidson, 1986, appendix III).

For the studies presented here on S. neumayeri, [.sup.14.C]-alanine was used as the radioactive tracer of rates of protein synthesis. The reasons for this choice are explained in the Results under the heading Validation of protocols. Experiments were conducted at -1.0 [degrees]C in 20-ml glass scintillation vials in a total volume of 14 ml at a concentration of about 1000 embryos or larvae per milliliter. The exact number of animals was always known and depended upon the developmental stage being studied.

Time-course experiments were conducted in which [.sup.14.C]-alanine was added to a final concentration of 9.0 mol [l.sup.-1] (Perkin Elmer, Wellesley, MA; 6.1 MBq [micro][mol.sup.-1]). These incubations lasted about 1.5 h, during which a series of samples was taken by placing 1 ml of suspended embryos or larvae onto a filter (pore size 8 [micro]m, Nucleopore). After a series of washings with cold seawater to remove unincorporated radioactivity, each filter containing embryos or larvae was frozen in liquid nitrogen. Subsequently, the following were measured: (1) rates of alanine transport, (2) change in intracellular specific activity of alanine in the precursor free amino acid pool, and (3) incorporation of radioactive alanine into protein. The amount of alanine transported by embryos or larvae was determined as described previously for developing sea urchins (Pace and Manahan, 2006). Reversed-phase high-performance liquid chromatography was used to separate and quantify the different amino acids in the embryos and larvae of the ethanol (70%)-extracted free amino acid pool (see Fig. 1D). Alanine was quantified through peak area comparison with standards of known concentration. The radioactivity in the alanine peak was determined by liquid scintillation counting, after collection of chromatographic eluent fractions (see Fig. 1E). The amount of [.sup.14.C]-alanine incorporated into protein was measured by precipitation with cold 5% trichloroacetic acid. Each precipitate was collected on a GF/C filter, and the radioactivity was counted using Scintisafe (Fisher) scintillation cocktail in a [.sup.14.C]-quench-corrected Beckman liquid scintillation counter.

The change with time in specific activity of [.sup.14.C]-alanine in the intracellular free amino acid pool was used to convert measurements of [.sup.14.C]-alanine in protein to rates of total alanine incorporation into protein. As expected under our experimental conditions, the specific activity of [.sup.14.C]-alanine in the intracellular free amino acid pool increased predictably with time of exposure of embryos (or larvae) to the radioactive substrate in seawater (Fig. 1A). Calculation of the specific-activity-corrected total amount of alanine incorporated into protein (measured as [.sup.14.C]-alanine in each sample of tricarboxylic acid precipitate and the corresponding change in specific activity) was based on the average specific activity of [.sup.14.C]-alanine for each time interval sampled. The amount (total moles of both radioactive and nonradioactive alanine) of alanine incorporated into protein was then converted to units of protein mass (Fig. 1C) on the basis of the amino acid composition of protein in S. neumayeri (from Marsh et al., 2001). Alanine is 6.6% of the amino acids that constitute protein in embryos and larvae of S. neumayeri (average molecular weight of protein amino acids is 142.1 g [mol.sup.-1]: Marsh et al., 2001). Figure 1C shows the results of such calculations and conversions to measure a protein synthesis rate based upon changing specific activity (Fig. 1A), the incorporation into protein of [.sup.14.C]-alanine (Fig. 1B), and the mole-percent alanine in developmental stages of S. neumayeri (6.6%).

Rates of protein synthesis were converted to protein-specific fractional rates of synthesis by standardizing absolute synthesis rates to the amount of total protein content present in the stages of development studied. Total protein was quantified using the Bradford assay (Bradford, 1976), modified for use with small tissue samples in embryos and larvae (Jaeckle and Manahan, 1989).

Rates of oxygen consumption

Rates of oxygen consumption were measured using microbiological oxygen demand ([micro]BOD) vials (details in Marsh and Manahan, 1999). Each [micro]BOD vial contained oxygen-saturated seawater, and a micropipette was used to place embryos or larvae into the vial. Numbers of individuals ranged from 200-500 per vial, depending on developmental stage. All respiration measurements were conducted at -1.0 [degrees]C for 5 h. For each developmental stage studied, six replicate measurements of respiration were made. Control vials with no animals present were set up in parallel to correct for possible background respiration. At the end of each incubation, vials were gently opened and a sample of the seawater (500 [micro]l) was removed with a gas-tight syringe and injected into a polarographic oxygen sensor housed in a water jacket (Strathkelvin, RC-100) equilibrated to -1.0 [degrees]C. Animals were then removed from each vial and enumerated under a microscope to ensure an accurate count of embryos or larvae responsible for each respiratory measurement. Rates of oxygen consumption are presented as averages [+ or -] standard error in units of pmol [O.sub.2] individua[l.sup.-1] [h.sup.-1].


Cost of protein synthesis

Experiments to determine the cost of protein synthesis in embryos and larvae of S. neumayeri were conducted by quantifying the difference in protein synthesis and oxygen consumption rates in the presence and absence of the inhibitors anisomycin and emetine (Sigma Chemical Co.). Different concentrations of anisomycin were tested for potential nonspecific effects on physiology. Oxygen consumption rates were converted to energy units using an oxyenthalpic value of 484 kJ mol [O.sub.2.sup.-1] (Gnaiger, 1983). Costs of protein synthesis are given as joules (mg protein synthesized)[.sup.-1].


Validation of protocols

Protein synthesis measurements. In the experiments reported here, alanine was selected as the radioactive tracer to measure rates of protein synthesis because it is rapidly taken up from seawater by Antarctic sea urchin embryos and larvae (Table 1) and is quantifiable in the intracellular free amino acid pool (required for measuring intracellular specific activity of [.sup.14.C]-alanine: Fig. 1A, D). The fact that alanine is not an essential amino acid and might be rapidly converted into other [.sup.14.C]-labeled amino acids is a potential source of error in protein synthesis rates measured on the basis of [.sup.14.C]-alanine as the precursor. Our results, however, showed no interconversion of [.sup.14.C]-alanine to other [.sup.14.C]-labeled amino acids during the course of our measurements: the time series of radio-chromatograms shown in Figure 1E illustrates that the [.sup.14.C]-radioactivity in the intracellular free amino acid pool was determined to be [.sup.14.C]-alanine. The specific activity of [.sup.14.C]-alanine in the intracellular free amino acid pool increased linearly, as expected, with increasing exposure of embryos to [.sup.14.C]-alanine in seawater (Fig. 1A). Similarly, a linear increase occurred in the rate of incorporation of [.sup.14.C]-alanine into protein (Fig. 1B). Combined, the data given in Figure 1A-E illustrate that [.sup.14.C]-alanine can be used for accurate measurements of protein synthesis rates at -1.0 [degrees]C for embryos and larvae of the Antarctic sea urchin S. neumayeri.

Use of protein synthesis inhibitors. The costs of protein synthesis were measured with specific inhibitors of protein synthesis, known from previous studies to be effective in marine invertebrates (Fenteany and Morse, 1993; Ioannou et al., 1998; Pace and Manahan, 2006). For our studies with the Antarctic sea urchin, we found that anisomysin has the advantage over other potential inhibitors of being easily soluble in cold seawater (-1.0 [degrees]C). Furthermore, results of experiments to define the optimal concentration of inhibitor (Figs. 2, 3, 4) showed that, with our protocols, anisomysin effectively inhibited protein synthesis at low concentrations (10 [micro]mol [l.sup.-1]) with no measurable side effects. Regarding possible side effects of this inhibitor, anisomycin at 10 [micro]mol [l.sup.-1] did not decrease rates of [.sup.14.C]-alanine transport by blastulae, gastrulae, and larvae (Table 1). Even at concentrations up to 100 [micro]mol [l.sup.-1], anisomycin did not decrease rates of [.sup.14.C]-alanine transport in gastrulae (Fig. 3C). Importantly, anisomycin did not differentially affect respiration relative to protein synthesis--a key verification of its specificity in our experimental conditions. At the lowest anisomycin concentration tested (5 [micro]mol [l.sup.-1]), protein synthesis decreased 78% in blastulae (Fig. 2A) and 56% in gastrulae (Fig. 3A). When the concentration of anisomycin was increased 20-fold, to 100 [micro]mol [l.sup.-1], further inhibition of protein synthesis was less than about 20% relative to the rate at 5 [micro]mol [l.sup.-1] (Figs. 2A and 3A). Inhibition of respiration rates showed a similar trend to that observed for protein synthesis (blastulae: Fig. 2B; gastrulae: Fig. 3B). For blastulae and gastrulae, proportional decreases over a range of anisomysin concentrations were measured for rates of both respiration and protein synthesis. This resulted in calculations of similarly low costs of protein synthesis within each developmental stage tested, at 0.24 [+ or -] 0.03 (SE) J (mg protein synthesized)[.sup.-1] for blastulae (Fig. 4A) and 0.64 [+ or -] 0.05 J (mg protein synthesized)[.sup.-1] for gastrulae (Fig. 4B). Nonspecific effects of anisomysin would most likely be seen as further decreases in respiration rate (e.g., the drug impacting some other metabolic process) with no corresponding decrease in protein synthesis rate, thereby causing a significant artifactual increase in the calculated cost of protein synthesis. As these nonspecific effects were not seen in our experiments for measurements of amino acid transport, respiration, and protein synthesis, we conclude that anisomysin is an effective and specific inhibitor of protein synthesis in developing stages of the Antarctic sea urchin S. neumayeri.




Fractional rates of protein synthesis

Rates of protein synthesis were high for cultures of S. neumayeri developing at -1 [degrees]C. Table 2 gives the fractional rates of protein synthesis calculated from total protein content and synthesis rates for embryos and larvae. Embryos and early larval stages (19-day-old, unfed) had protein synthesis rates of 4.1% [+ or -] 0.24% individua[l.sup.-1] [h.sup.-1]. This rate increased at gastrulation and ranged (n = 2) from 7.0% to 7.2% embry[o.sup.-1] [h.sup.-1] in 10-day-old gastrulae. In later larval stages that were unfed for 43 days, fractional rates of synthesis decreased and ranged (n = 2) from 0.4% to 0.5% larv[a.sup.-1] [h.sup.-1].

Stage- and age-specific costs of protein synthesis

The experiments to define the optimal concentrations of the inhibitor anisomysin (Figs. 2, 3, and 4) revealed that the costs of protein synthesis in Antarctic sea urchins, at less than 1 J (mg protein synthesized)[.sup.-1], are low in comparison to those of other animals studied (as reviewed in the Discussion section). Detailed studies of rates and costs of protein synthesis in blastulae, gastrulae, and larvae were undertaken for several different-aged stages of development and cohorts of such stages spawned from different adults. Figure 5 shows rates of protein synthesis for two cohorts of blastulae. Rates of protein synthesis with no inhibitor (control) for these two cohorts were 5.9 [+ or -] 0.4 (SE) and 8.9 [+ or -] 0.9 ng protein embry[o.sup.-1] [h.sup.-1] for 6- and 7-day-old blastulae, respectively. Rates of protein synthesis decreased to 1.4 [+ or -] 0.2 and 3.1 [+ or -] 0.6 ng protein embry[o.sup.-1] [h.sup.-1] in the presence of 10 [micro]mol [l.sup.-1] anisomycin. This equates to a decrease in protein synthesis rates of 76% and 65%. Rates of respiration in the absence of inhibitor were 13.1 [+ or -] 0.2 and 11.3 [+ or -] 0.4 pmol [O.sub.2] embry[o.sup.-1] [h.sup.-1] for 6- and 7-day-old blastulae, respectively. In the presence of 10 [micro]mol [l.sup.-1] anisomycin, these respiration rates decreased to 63% and 65% of non-inhibited rates (with inhibitor: 8.3 [+ or -] 0.1 and 7.3 [+ or -] 0.2 pmol [O.sub.2] embry[o.sup.-1] [h.sup.-1] for 6- and 7-day-old blastulae, respectively). Based on the differences in these rates of synthesis and respiration, the costs of protein synthesis were 0.52 and 0.33 J (mg protein synthesized)[.sup.-1] for 6- and 7-day-old blastulae, respectively.

Costs of protein synthesis were further examined in cohorts of gastrulae of different ages and parents from those used to test the potential concentration dependency of anisomycin on costs of protein synthesis (Fig. 4B). Figure 6 shows the rates of protein synthesis and respiration in gastrulae, with and without inhibitor. For these three different experiments, costs of protein synthesis were 0.27, 0.13, and 0.25 J (mg protein synthesized)[.sup.-1]. For 19-day-old larvae (Fig. 7) the cost of protein synthesis calculated using anisomysin as the inhibitor was 0.48 J (mg protein synthesized)[.sup.-1]. This value is similar to the cost determined with a second inhibitor of protein synthesis: emetine at 500 [micro]mol [l.sup.-1] resulted in a cost of protein synthesis of 0.52 J (mg protein synthesized)[.sup.-1]. An additional experiment (data not shown) using anisomycin on a different cohort of 19-day-old larvae gave a cost of protein synthesis of 0.51 J (mg protein synthesized)[.sup.-1]. These similar results for costs of synthesis, obtained with two different inhibitors and different cohorts of larvae, are a further verification that costs of protein synthesis in the Antarctic sea urchin are low.


Empirical and theoretical costs of protein synthesis in the Antarctic sea urchin

Direct measurements of the cost of protein synthesis in embryos and larvae of the Antarctic sea urchin ranged from 0.13 to 0.79 J (mg protein synthesized)[.sup.-1] (Fig. 8A). The average empirical cost of synthesis of 0.41 [+ or -] 0.05 J (mg protein synthesized)[.sup.-1] (n = 16) is compared with theoretical costs of synthesis--3 J (mg protein synthesized)[.sup.-1]--in Figure 8B. Values ranging from 3 to 8 J [mg.sup.-1] have been considered representative of the theoretical cost of protein synthesis based on stoichiometric considerations (Buttery and Boorman, 1976; Waterlow et al., 1978; Aoyagi et al., 1988; Houlihan, 1991; Smith and Houlihan, 1995; Fraser et al., 2002; Smith and Ottema, 2006). For the Antarctic sea urchin, we have calculated a theoretical minimum cost to be 0.86 J (mg protein synthesized)[.sup.-1]. To calculate the theoretical costs, we used the generally agreed on minimum value of 4 ATP equivalents per peptide bond formed (Buttery and Boorman, 1976; Aoyagi et al., 1988; Smith and Houlihan, 1995). Previous work in our laboratory measured the amino acid composition of protein in eggs, blastulae, gastrulae, and early larval stages of S. neumayeri and calculated an average molecular weight of 142.1 [+ or -] 0.78 (SE) grams per mole of amino acid in protein (Marsh et al., 2001). For our calculation of the theoretical minimum cost of 0.86 J (mg protein synthesized)[.sup.-1], we used the measured average molecular weight of protein amino acids in S. neumayeri and the well-accepted value of the energy of ATP hydrolysis of 30.5 kJ (mol ATP)[.sup.-1] (Lehninger, 1975, pp. 402-403; Alberts et al., 1994, p. 670; Stryer, 1995, pp. 445-446). The theoretical cost of protein synthesis in S. neumayeri is calculated as follows:




ATP requirement (mole ATP per mole peptide bond synthesized) = 4 mole ATP per mole amino acid

Conversion of moles ATP to joules of energy (kJ energy per mole ATP) = 4 mole ATP per mole amino acid X 30.5 kJ per mole ATP (total energy cost = 122 kJ per mole amino acid)

Conversion to energy cost of protein synthesis = 122 kJ per mole amino acid / 142.1 grams protein per mole amino acid = 0.86 kJ (g protein synthesized)[.sup.-1]

Theoretical cost = 0.86 J (mg protein synthesized)[.sup.-1]

A free energy value of 50 kJ (mol ATP)[.sup.-1] can also be used to calculate a higher-end theoretical value of protein synthesis costs in S. neumayeri. This is a value also used for ATP equivalents and is based on assumptions of the cellular environment in which free energy reactions are sensitive to temperature, pH, concentrations of [Mg.sup.+2], concentrations of ATP and ADP, etc. (Lehninger, 1975; Alberts et al., 1994; Stryer, 1995). A value of 50 kJ (mol ATP)[.sup.-1] would give a theoretical cost of 1.41 J (mg protein synthesized)[.sup.-1]. In summary, these calculations based on use of 4 ATP per peptide bond show that theoretical costs of protein synthesis in the Antarctic sea urchin are approximately 1 J (mg protein synthesized)[.sup.-1]. Our empirically determined costs averaged 0.41 [+ or -] 0.05 J (mg protein synthesized)[.sup.-1] (Fig. 8B). Empirical and theoretical costs of protein synthesis in the Antarctic sea urchin are further considered in the Discussion.


Sea urchin embryos from temperate regions have long been used in developmental biology for studies of molecular biology, biochemistry, physiology, and other aspects of animal development (reviewed in Davidson, 1986; Sea Urchin Genome Sequencing Consortium, 2006). In the current study we build upon the experimental tractability of sea urchins to understand developmental physiology in the cold and, specifically, to define the biochemical bases for energy costs of early animal development in such environments. We measured rates of protein synthesis during development of the Antarctic sea urchin Sterechinus neumayeri and the metabolic demands of such synthesis. Our major conclusions are summarized in Table 2 and Figure 8.

First, rates of protein synthesis are remarkably high in this Antarctic species, with mean fractional rates of protein synthesis ranging from 0.5% to 7.1% [h.sup.-1] in different stages developing at -1 [degrees]C (Table 2). These fractional rates of protein synthesis in the cold are in the same range as or higher than those reported for temperate species of sea urchin developing at much higher temperatures (e.g., at 15 [degrees]C, Lytechinus pictus = 0.6% [h.sup.-1] [Pace and Manahan, 2006]; at 16 [degrees]C, Strongylocentrotus purpuratus = 1.1% [h.sup.-1] [Goustin and Wilt, 1981]; at 25 [degrees]C, Arbacia punctulata = 1.9% [h.sup.-1] [Fry and Gross, 1970]). "Tracer dose" methods similar to those used in the current study have revealed high fractional rates of protein synthesis of >1% [h.sup.-1] in gastropod larvae (abalone Haliotis rufescens: Vavra and Manahan, 1999). High fractional rates measured with "flooding dose" methods have been reported by McMillan and Houlihan (1988) in fish liver preparations (>1% [h.sup.-1]) and by Conceicao et al. (1997) in fish larvae (~6% [h.sup.-1]). High fractional rates of protein synthesis appear to be a common occurrence for developmental stages of several animal species studied. Such high rates are consistently found, whether flooding dose or tracer dose methods are employed. Both methods are equally valid and the choice of method is species-dependent. For example, radiolabeled tracer amounts are appropriate for organisms with large intracellular pools of free amino acid.

The second major finding of this study is that the energetic cost of maintaining high rates of protein synthesis in the cold is strikingly and consistently low (Fig. 8A). For 16 experiments spanning blastula to larval stages, the cost of protein synthesis averaged 0.41 [+ or -] 0.05 (SE) J (mg protein synthesized)[.sup.-1]. Although rates of protein synthesis were highly variable depending on developmental stage (6-14 ng protein [h.sup.-1], Figs. 5A and 6A), we found no evidence that cost of protein synthesis varied in S. neumayeri as a function of rate of synthesis. Costs of protein synthesis in this Antarctic sea urchin are fixed, like those we reported for a temperate species of sea urchin (Pace and Manahan, 2006). A value of 0.41 J (mg protein synthesized)[.sup.-1] is a uniquely low cost of protein synthesis in the cold, given that costs for most animals studied average around 8 J (mg protein synthesized)[.sup.-1]. For example, our previous study of a temperate species of sea urchin, Lytechinus pictus, measured the cost of protein synthesis to be 8.4 J (mg protein synthesized)[.sup.-1] (Pace and Manahan, 2006). Reports for other species are similarly high: birds at 5.4 J [mg.sup.-1] (Aoyagi et al., 1988); codfish at 8.7 J [mg.sup.-1] (Lyndon et al., 1989); and the mussel Mytilus edulis at 11.4 J [mg.sup.-1] (Hawkins et al., 1989).

Given the high rate of protein synthesis measured in Antarctic sea urchin embryos, the metabolic cost of that synthesis has to be low to be compatible with the known respiration rate of this species (respiration values from this study; see also Shilling and Manahan, 1994; Marsh and Manahan, 1999; Marsh et al., 1999). Costs of protein synthesis during development of Antarctic sea urchin embryos and larvae, and the energy allocation required for such rates, are illustrated as "metabolic pie-charts" in Figure 9. Using the average cost of protein synthesis of 0.41 J (mg protein synthesized)[.sup.-1] from Figure 8B, rates of synthesis were converted into energy equivalents to determine what percentage of metabolism was required to support protein synthesis. This analysis shows the metabolic significance of protein synthesis, especially in early embryonic development when protein synthesis alone can account for over 50% of metabolism. Gastrulae, for instance, had a very high (~7% [h.sup.-1]) fractional rate of protein synthesis (Table 2). The cost of this synthesis must be low given that, even at a synthesis cost of 0.41 J (mg protein synthesized)[.sup.-1], 54% of metabolic energy expenditure was accounted for by the protein synthesis rate.

While it is important to understand the metabolic cost of protein synthesis, it is also critical to put this cost into the context of other energy-requiring processes contributing to the metabolic rate of an organism (Hochachka, 1988; Leong and Manahan, 1997, 1999; Marsh et al., 2001; Fraser et al., 2002). For the developmental stages of the Antarctic sea urchin, the costs of maintaining ion gradients, another metabolically expensive process, have to be accounted for in addition to the high rates of protein synthesis being supported. The sodium pump ([Na.sup.+],[K.sup.+]-ATPase) is essential for maintaining specific ion gradients that, in turn, are used to drive active transport of organic substrates such as amino acids (Christensen et al., 1967). The physiologically active fraction of the [Na.sup.+],[K.sup.+]-ATPase enzyme (the percentage of total sodium pump active in vivo) has been measured during development of this species of Antarctic sea urchin (Leong and Manahan, 1999). Combining data on the metabolic cost of protein synthesis and the metabolic cost of the sodium pump now permits a better quantification of the biochemical bases of the "cost of living" for developing stages of S. neumayeri at -1 [degrees]C. The physiologically active fraction of the sodium pump accounted for 12% of metabolism in blastulae and gastrulae. This fraction of metabolism increased to 20% for early larval stages and was the dominant energy-requiring process in later larval stages, when the physiologically active fraction of this single enzyme accounted for 84% of total metabolic energy (Leong and Manahan, 1999). These results on the sodium pump, when combined with the analysis in the current study of metabolic cost of protein synthesis for the same stages of development, allow for the biochemical definition of processes responsible for between 64% and 87% of metabolism in this Antarctic sea urchin (Fig. 9). These two major processes accounted for, on average, approximately 75% of the metabolic rate, and their relative proportion changed during development. In gastrulae, sodium pump activity accounted for only 12% of metabolism. High fractional protein synthesis rates of 7% [h.sup.-1] can be accommodated at 54% of metabolism, but only because the cost of synthesis is low (Fig. 8A). In later larval stages (6-weeks-old) the reverse situation can also be accommodated metabolically, when the dominant component is the physiologically active fraction of the sodium pump accounting for 84% of metabolism (Leong and Manahan, 1999). This leaves only 16% of metabolism to accommodate all other costs of living in larvae. Again, the low cost of protein synthesis in this Antarctic species permits protein synthesis in larval stages to fit into this biological constraint of 16%, as it accounts for only 3% of metabolism in larvae (Fig. 9).


The proportion of total metabolism accounted for by protein synthesis is remarkably similar in Antarctic and temperate species of sea urchins. In blastulae of S. neumayeri, protein synthesis accounted for 52% of metabolic rate at -1 [degrees]C, a value essentially identical to that of blastulae of L. pictus developing at 15 [degrees]C (54% for protein synthesis). A similar proportionality of metabolic cost of protein synthesis is evident in gastrulae of these Antarctic and temperature sea urchin species. Values of about 50% of metabolism attributed to protein synthesis are common in the literature (e.g., Wells et al., 1983; Houlihan et al., 1988). A value of 50% can only be explained by a very low cost of synthesis, given the high rates of synthesis in Antarctic sea urchin embryos.

The cost of protein synthesis of 0.41 [+ or -] 0.05 J (mg protein synthesized)[.sup.-1] determined in the current study is based on direct measurements using two different inhibitors of protein synthesis. Anisomycin reversibly blocks the transfer of amino acid-tRNAs into the A-site of ribosomes (Pestka, 1971; Kinzy et al., 2002). Emetine has a different action and irreversibly stabilizes ribosomes onto mRNA transcripts (Pestka, 1971). The low values we report here for protein synthesis costs measured using both of these inhibitors confirm the previous findings of Marsh et al. (2001) of 0.45 J (mg protein synthesized)[.sup.-1]. This latter value was calculated by a different method for measuring protein synthesis costs--the "correlative" approach that is based on a linear regression analysis of the relationship between the change of oxygen consumption and protein synthesis rates. This agreement between the direct inhibitor and correlative approaches for measuring the costs of protein synthesis has also been observed in developmental stages of a temperate species of sea urchin (Pace and Manahan, 2006). The high rates of protein synthesis presented in the current study support previous measurements of macromolecular synthesis rates during development of the Antarctic sea urchin S. neumayeri. Marsh et al. (2001) reported that rates of total RNA synthesis were elevated in embryonic stages, with rates similar to those measured in temperate echinoderms. Rates of mRNA synthesis were about 4 times higher in S. neumayeri at -1.5 [degrees]C than were rates in temperate sea urchin embryos (at 16-19 [degrees]C). Using both essential (leucine) and non-essential (alanine) amino acids as tracers to measure protein synthesis rates, Marsh et al. (2001) also reported high fractional rates of protein synthesis in developing Antarctic sea urchins. Similarly, the amino acid chosen to measure rates of protein synthesis for mammals is unimportant since different amino acids (glycine, lysine, and tyrosine) gave very similar results in liver and muscle (Waterlow et al., 1978, chapter 10, p. 361). This general finding that many different amino acid classes can be used to measure protein synthesis is in accord with other studies of protein synthesis in sea urchins summarized in Regier and Kafatos (1977). The fractional rates of protein synthesis we report here for the Antarctic sea urchin (Table 2), measured with [.sup.14.C]-alanine, fall within the range reported by Marsh et al. (2001).

Costs of protein synthesis in adult stages of some Antarctic marine invertebrates are not as low as we report here for developmental stages of Antarctic sea urchins. Whitely et al. (1996) measured high costs of protein synthesis in conjunction with low protein synthesis rates in the Antarctic isopod Glyptonotus antarcticus. Storch and Portner (2003) addressed the question of temperature-dependent costs of protein synthesis in cell-free protein synthesis extracts of gill tissue from European and Antarctic scallops and reported no difference between these two species. The significant differences in the costs of protein synthesis between embryos and larvae of the Antarctic sea urchin and these other adult species may be the result of a unique physiology that accompanies development in extreme-cold environments. The physiological requirements of early development, with small energetic capacity and the need for rapid cellular growth, have led to suggestions that the costs of growth might be lower for early life-history stages than for adults (Wieser et al., 1988; Rombough, 1994). Such an explanation may, in part, explain the low cost of protein synthesis for developmental stages of Antarctic echinoderms. Nonetheless, this does not provide a mechanistic explanation for the very low cost of protein synthesis we report here (Fig. 8A), which we discuss below.

Calculations of theoretical costs of protein synthesis involve a suite of assumptions that can create a range of values. A minimum theoretical cost of protein synthesis routinely reported in the literature is 3 J (mg protein synthesized)[.sup.-1] (Aoyagi et al., 1988; Lyndon et al., 1989; Hawkins et al., 1989; Houlihan et al., 1990). This value is based upon an average molecular weight of 110 grams per mole amino acid in protein and 75.5 kJ (mol ATP)[.sup.-1] (e.g., Buttery and Boorman, 1976; Waterlow et al., 1978; Aoyagi et al., 1988). This energy that is theoretically available from ATP hydrolysis is predicated upon complete oxidation of glucose to C[O.sub.2] and water, and application of the energy released to the formation of high-energy phosphate bonds. It is noteworthy that the value of 75.5 kJ (mol ATP)[.sup.-1] calculated from these reactions assumes 100% energy transfer efficiency, a situation that is unlikely in a biochemical process. Much of the variance in estimates of theoretical costs of protein synthesis comes from assumptions about the average molecular weight of amino acids in protein of different species and from the conversion of ATP energy equivalents from the 4 ATP required for peptide bond synthesis. For instance, the measured amino acid composition of protein of S. neumayeri is 142.1 g [mol.sup.-1], which is about 30% higher than the typically used value of 110 g [mol.sup.-1] and would result in about a 30% lower cost of synthesizing a unit mass of protein in S. neumayeri. In addition, the energy of ATP hydrolysis (not ATP formation) under standard conditions of 30.5 kJ [mol.sup.-1] (Lehninger, 1975) might more reasonably be used to calculate protein synthesis costs. Combining measured values of amino acid composition in protein with 30.5 kJ (mol ATP)[.sup.-1] yields a theoretical minimum cost of protein synthesis in the Antarctic sea urchin of 0.86 J (mg protein synthesized)[.sup.-1] (Fig. 8B). This theoretical cost would be 1.41 J (mg protein synthesized)[.sup.-1] if the calculation were based upon 50 kJ (mol ATP)[.sup.-1], a value commonly cited for the energy of ATP equivalents in cellular environments (Lehninger, 1975). Our calculations show that costs of protein synthesis of about 1 J (mg protein synthesized)[.sup.-1] are theoretically possible for this Antarctic sea urchin. Our direct measurements of the cost of protein synthesis during development of this species all support empirical values less than 1 J (mg protein synthesized)[.sup.-1] (Fig. 8A).

Although there is not a perfect agreement between theoretical and empirical costs of protein synthesis in our study, it is important to emphasize how little is understood about cellular conditions that might influence energy availability from ATP. This is particularly the case for embryos developing in extreme-cold Antarctic conditions. Further studies of energy use during early animal development in the cold may reveal unique possibilities for enhanced efficiency of cellular metabolism in subzero seawater temperatures. Some possibilities might include increases in energy made available from hydrolysis of ATP or decreases in the number of ATP molecules required for protein synthesis in Antarctic embryos. Many different and diverse cellular processes contribute to estimates of the cost of protein synthesis when measurements are made in vivo. Some of these processes are likely to include cellular activities that are not directly associated with protein synthesis, such as energy requirements for amino acid transport and the translocation, folding, and degradation of proteins. Molecular chaperones are also known to require ATP (Fink, 1999), as do ubiquitin-mediated pathways for protein degradation (Hershko, 1988). In mammalian cells, for instance, up to 30% of newly synthesized proteins are defective ribosomal products that are immediately degraded--a process that requires substantial amounts of ATP (Schubert et al., 2000). Under different environmental conditions, variations in the relative contribution of such biological processes to metabolism could provide further insights into the mechanisms that set the low costs of protein synthesis measured for developmental stages of Antarctic sea urchins (Marsh et al., 2001; this study). Several hypotheses that might account for low costs of protein synthesis could be tested directly with cell-free, protein translation lysates. Temperature could easily be manipulated in such experiments, in combination with the ability to control for many of the aforementioned processes related to in vivo measurements of protein synthesis (e.g., elimination of costs of amino acid transport in cell-free preparations). We are currently investigating some of these possibilities using cell-free, protein translation lysates prepared from Antarctic sea urchin embryos.

In conclusion, developmental stages of animals generally require high rates of protein synthesis and turnover. At typical costs of protein synthesis, these rates could not be supported metabolically in the Antarctic sea urchin. Such rates are, however, possible in this species because of a low synthesis cost, allowing for low rates of respiration. The low cost of synthesis we report here opens the possibility that other unique physiological processes might exist and highlights the need for further studies of the biological mechanisms by which growth and development occur in extreme-cold environments.


We thank David Ginsburg, Allison Green, Amanda Haag, and Michael Moore for assistance with rearing embryos and larvae over many months while in Antarctica. Emi Yamaguchi assisted with Bradford protein assays. Dr. Robert Maxson provided helpful discussions and advice. We also thank the Crary Laboratory staff at McMurdo Station, Antarctica, and Raytheon Polar Services for support. Our special thanks to Rob Robbins for exceptional support for animal collection (scuba diving) and field logistics while in Antarctica. This work was supported by the National Science Foundation under Grant No. 01-30398.

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Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371

Received 10 July 2006; accepted 3 January 2007.

* To whom correspondence should be addressed. E-mail:
Table 1 Alanine transport rates by embryos and larvae of Sterechinus
neumayeri with and without the addition of the protein synthesis
inhibitor anisomycin (10 [micro]mol [l.sup.-1] concentration)

                                   Transport rate
                                   (pmol Ala
Stage      Age (days)  Treatment   [h.sup.-1])          ANOVA (P value)

Blastulae   6          Control     0.52 [+ or -] 0.05   [0.46.sup.ns]
                       Anisomycin  0.58 [+ or -] 0.07
Blastulae   7          Control     1.20 [+ or -] 0.06   [0.66.sup.ns]
                       Anisomycin  1.13 [+ or -] 0.14
Gastrulae  10          Control     1.57 [+ or -] 0.13   [0.87.sup.ns]
                       Anisomycin  1.54 [+ or -] 0.09
Gastrulae  10          Control     1.42 [+ or -] 0.06   [0.92.sup.ns]
                       Anisomycin  1.44 [+ or -] 0.11
Gastrulae  14          Control     1.48 [+ or -] 0.05   [0.63.sup.ns]
                       Anisomycin  1.42 [+ or -] 0.11
Larvae     19          Control     1.74 [+ or -] 0.16   [0.60.sup.ns]
                       Anisomycin  1.64 [+ or -] 0.11

Embryonic stages listed above more than once (e.g., 2 experiments with
10-day-old gastrulae) represent different cohorts of embryos obtained
from spawning different parents. Errors of transport rates represent
standard error of the slopes of the increase of alanine with time. P
values are the significance levels for ANOVA analysis of the combined
regressions of transport rates with and without inhibitor for each
specific developmental stage (e.g., comparison of 6-day-old blastulae
with and without anisomycin); "ns" indicates no statistically
significant difference in transport rates.

Table 2 Protein content and fractional rates of protein synthesis in
embryos and larvae of the Antarctic sea urchin Sterechinus neumayeri

                                             Fractional protein
                   Protein content           synthesis rate
Developmental      [+ or -] SEM              [+ or -] SEM (% of
stage           n  (ng individua[l.sup.-1])  protein content [h.sup.-1])

Embryos and     4  203.1 [+ or -] 12.7       4.1% [+ or -] 0.24%
  early larvae
Gastrulae (10-  2  199.7 [+ or -] 0.8        7.1% [+ or -] 0.10%
Larvae (unfed   2  179.5 [+ or -] 4.5        0.5% [+ or -] 0.05%
  for 43 days)
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Author:Pace, Douglas A.; Manahan, Donal T.
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Date:Apr 1, 2007
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