Experimental ocean acidification alters the allocation of metabolic energy

Abstract

Energy is required to maintain physiological homeostasis in response to environmental change. Although responses to environmental stressors frequently are assumed to involve high metabolic costs, the biochemical bases of actual energy demands are rarely quantified. We studied the impact of a near-future scenario of ocean acidification [800 µatm partial pressure of CO2 (pCO2)] during the development and growth of an important model organism in developmental and environmental biology, the sea urchin Strongylocentrotus purpuratus. Size, metabolic rate, biochemical content, and gene expression were not different in larvae growing under control and seawater acidification treatments. Measurements limited to those levels of biological analysis did not reveal the biochemical mechanisms of response to ocean acidification that occurred at the cellular level. In vivo rates of protein synthesis and ion transport increased ∼50% under acidification. Importantly, the in vivo physiological increases in ion transport were not predicted from total enzyme activity or gene expression. Under acidification, the increased rates of protein synthesis and ion transport that were sustained in growing larvae collectively accounted for the majority of available ATP (84%). In contrast, embryos and prefeeding and unfed larvae in control treatments allocated on average only 40% of ATP to these same two processes. Understanding the biochemical strategies for accommodating increases in metabolic energy demand and their biological limitations can serve as a quantitative basis for assessing sublethal effects of global change. Variation in the ability to allocate ATP differentially among essential functions may be a key basis of resilience to ocean acidification and other compounding environmental stressors.

Keywords: development; energetics; metabolic allocation; ocean acidification; sea urchin.

Fig. 1.

Size (A) and metabolic rate (B) in developing sea urchins under control (closed symbols) and seawater acidification (open symbols) treatments. (A) Changes in diameters of embryos (inverted triangles), body lengths of larvae (circles) and unfed larvae (triangles). Each data point represents mean ± SEM (n = 50 individuals). Where not visible, error bars fall within the graphical representation of the data point. For visual clarity, data points for a given x-axis value are slightly offset when symbols overlap. Body lengths did not differ between control and acidification treatments for fed (ANCOVA, P = 0.095, n = 2,081) and unfed (P = 0.537, n = 481) larvae. (B) Metabolic rate (measured as O₂ consumption) per individual as a function of body length. Error bars represent 1 SEM, n = 8–10 respiration assays. (Inset) Bar graph shows replicate measurements on 6-, 8-, and 10-d-old unfed larvae under control (Ctrl) and acidification (CO₂) treatments. Metabolic rates did not differ significantly between control and acidification treatments for fed larvae (ANCOVA, P = 0.326, n = 255). Metabolic rates of embryos and unfed larvae were elevated under acidification (ANOVA, P < 0.001, n = 97).

Fig. 2.

Protein content and synthesis in developing sea urchins under control (closed symbols) and seawater acidification (open symbols) treatments. (A) Protein content as a function of body length in embryos (triangles) and fed larvae (circles). Error bars indicate 1 SEM, n = 4–5 protein assays. Where not visible, error bars fall within the graphical representation of the data point. No statistical differences in protein content were observed between control and acidification treatments (embryos and unfed larvae, ANOVA, P = 0.063, n = 49; feeding larvae, ANCOVA, P = 0.847, n = 49). (Inset) The bar graph shows replicate measurements on 6-, 8-, and 10-d-old unfed larvae. (B) Each data point represents a protein synthesis rate, calculated from the combined slope (± SE) of duplicate six-point time-course assays of the amount of protein synthesized, corrected for intracellular specific activity of ¹⁴C-alanine in the free amino acid pool. Size-specific protein synthesis rates were significantly greater under acidification in growing larvae (ANCOVA, P = 0.009, n = 20) and (Inset) in unfed larvae (Post hoc test, P < 0.001, n = 12). See Fig. S2 for equations for the regression lines shown here in A and B.

Fig. 3.

Energy cost of protein synthesis in developing sea urchins. (A) Protein synthesis of 1-d-old embryos in the absence (solid regression line, rate = 0.51 ± 0.032 nanograms per individual per hour, slope ± SE of slope) and presence (dashed regression line, rate = 0.03 ± 0.004 nanograms per individual per hour) of 100 µM emetine. Synthesis rates were calculated from triplicate, five-point time-course assays. (B) Metabolic rate measured as oxygen consumption, for the cohort of embryos in A, converted to energy equivalents (expressed in microJoules). Error bars represent 1 SEM, n = 8 respiration assays. (C) The cost of protein synthesis was calculated from the simultaneous decreases in protein synthesis and metabolic rate under inhibition by emetine. Error bars represent 1 SEM. The energy cost of protein synthesis did not differ significantly between control (black bar, n = 3) and seawater acidification (open bar, n = 3) treatments (t test, df = 4, t = 1.178, P = 0.304). The grand mean (hatched bar) of 2.4 ± 0.21 J/mg protein synthesized was calculated from all cost determinations (n = 9) for two pCO₂ treatments and developmental stages spanning 1-d-old embryos to 10-d-old larvae.

Fig. 4.

Ion transport rate, total enzyme activity, and gene expression of Na⁺,K⁺-ATPase in developing sea urchins. (A) In vivo Na⁺,K⁺-ATPase activity was determined from ⁸⁶Rb⁺ transport rates, corrected for the specific activity of K⁺ in seawater, in the absence (solid regression line, rate = 2.1 ± 0.19 picomoles K⁺ per individual per minute, slope ± SE of slope) and presence (dashed regression line, rate = 0.9 ± 0.18 picomoles K⁺ per individual per minute) of 2 mM ouabain. (B) In vivo Na⁺,K⁺-ATPase activity under control (closed symbols) and seawater acidification (open symbols) treatments in embryos (triangles) and larvae (circles) as a function of body size. Each data point was calculated from time-course assays as shown in A. (Inset) The bar graph shows duplicate measurements on 6- and 8-d-old unfed larvae. Seawater acidification treatment significantly increased in vivo Na⁺,K⁺-ATPase activity in growing larvae (ANCOVA, P = 0.016, n = 20) as well as in unfed larvae (Inset). Post hoc test, P = 0.001, n = 8. (C) Relative Na⁺,K⁺-ATPase gene expression and total enzyme activity. Error bars indicate 1 SEM, n = 3–4 assays. Where not visible, error bars fall within the graphical representation of the data point. No statistical differences in gene expression or total enzyme activity occurred between control and acidification treatments, with the exception of 4-d-old larvae (∼220 µm). For this stage, gene expression did not predict the changes in physiological rates of ion transport (B), because gene expression was higher in the control relative to acidification treatment (post hoc test, P < 0.001).

Fig. 5.

Changes in ATP allocation to protein synthesis (black), in vivo Na⁺,K⁺-ATPase activity (gray), and the unaccounted fraction of total ATP (white) in developing sea urchins. (A and B) Metabolic energy budgets for embryos, prefeeding, and fed larvae (A) and unfed larvae (B) under control (outlined in blue) and seawater acidification and CO₂ (outlined in red) treatments. Values within each pie chart indicate the proportion (%) of the total metabolic rate allocated to each category. Data used for calculation of ATP allocation are given in Table S4. (C) Images of 6-d-old fed and unfed larvae under control and acidification treatments. (Scale bars: 100 µm.)