Integrative biological analyses of responses to food deprivation reveal resilience mechanisms in sea urchin larvae.

A fundamental question in ecology is how organisms survive food deprivation. In the ocean, climate change is impacting the phenology of food availability for early life-history stages of animals. In this study, we undertook an integrative analysis of larvae of the sea urchin Strongylocentrotus purpuratus-an important keystone species in marine ecology and a molecular biological model organism in developmental biology. Specifically, to identify the mechanisms of resilience that maintain physiological state and the ability of organisms to recover from food deprivation, a suite of molecular biological, biochemical, physiological and whole organism measurements was completed. Previous studies focused on the importance of energy reserves to sustain larvae during periods of food deprivation. We show, however, that utilization of endogenous energy reserves only supplied 15% of the metabolic requirements of long-term survival (up to 22 days) in the absence of particulate food. This large energy gap was not supplied by larvae feeding on bacteria. Estimates of larval ability to transport dissolved organic matter directly from seawater showed that such substrates could fully supply metabolic needs. Integrative approaches allowed for filtering of gene expression signatures, linked with gene network analyses and measured biochemical and physiological traits, to identify biomarkers of resilience. We identified 14 biomarkers related to nutrition-responsive gene expression, of which a specific putative amino acid transporter gene was quantified in a single larva experiencing continuous nutritional stress. Advances in applications of gene expression technologies offer novel approaches to determine the physiological state of marine larval forms in ecological settings undergoing environmental change.

Thermal sensitivities of respiration and protein synthesis differ among larval families of the Pacific oyster, Crassostrea gigas.

Understanding the mechanisms of biological responses to environmental change is a central theme in comparative and evolutionary physiology. Here, we analyzed variation in physiological responses to temperature, using 21 full-sibling larval families of the Pacific oyster, Crassostrea gigas. Pedigrees were confirmed with genetic markers for adult broodstock obtained from our breeding program. From these 21 larval families, 41 determinations of thermal sensitivity (Q10 values) were assayed for larvae of different sizes. For respiration, thermal sensitivity was consistent within a larval family during growth, but showed significant differences among families. Different Q10 values were evident among 21 larval families, with family accounting for 87% of variation. Specifically, four larval families maintained an increased thermal sensitivity for respiration (Q10 of 3). This physiology would confer resilience to rising temperature by matching the increased energy demand of protein synthesis (Q10 of 3 previously reported). For protein synthesis, differences in Q10 values were also observed. Notably, a family was identified that had a decreased thermal sensitivity for protein synthesis (Q10 of 1.7 cf. Q10 of 3 for other families), conferring an optimal energy allocation with rising temperature. Different thermal sensitivities across families for respiration (energy supply) and protein synthesis (energy demand) were integrated into models of energy allocation at the whole-organism level. The outcome of these analyses provides insights into the physiological bases of optimal energy allocation with rising temperature. These transgenerational (egg-to-egg) experiments highlight approaches to dissect components of phenotypic variance to address long-standing questions of genetic adaptation and physiological resilience to environmental change.

Differing thermal sensitivities of physiological processes alter ATP allocation.

Changes in environmental temperature affect rate processes at all levels of biological organization. Yet the thermal sensitivity of specific physiological processes that affect allocation of the ATP pool within a species is less well understood. In this study of developmental stages of the Pacific oyster, Crassostrea gigas, thermal sensitivities were measured for growth, survivorship, protein synthesis, respiration and transport of amino acids and ions. At warmer temperatures, larvae grew faster but suffered increased mortality. An analysis of temperature sensitivity (Q10 values) revealed that protein synthesis, the major ATP-consuming process in larvae of C. gigas, is more sensitive to temperature change (Q10 value of 2.9±0.18) than metabolic rate (Q10 of 2.0±0.15). Ion transport by Na+/K+-ATPase measured in vivo has a Q10 value of 2.1±0.09. The corresponding value for glycine transport is 2.4±0.23. Differing thermal responses for protein synthesis and respiration result in a disproportional increase in the allocation of available ATP to protein synthesis with rising temperature. A bioenergetic model is presented illustrating how changes in growth and temperature affect allocation of the ATP pool. Over an environmentally relevant temperature range for this species, the proportion of the ATP pool allocated to protein synthesis increases from 35 to 65%. The greater energy demand to support protein synthesis with increasing temperature will compromise energy availability to support other essential physiological processes. Defining the trade-offs of ATP demand will provide insights into understanding the adaptive capacity of organisms to respond to various scenarios of environmental change.

Biochemical bases of growth variation during development: a study of protein turnover in pedigreed families of bivalve larvae (Crassostrea gigas).

Animal size is a highly variable trait regulated by complex interactions between biological and environmental processes. Despite the importance of understanding the mechanistic bases of growth, predicting size variation in early stages of development remains challenging. Pedigreed lines of the Pacific oyster (Crassostrea gigas) were crossed to produce contrasting growth phenotypes to analyze the metabolic bases of growth variation in larval stages. Under controlled environmental conditions, substantial growth variation of up to 430% in shell length occurred among 12 larval families. Protein was the major biochemical constituent in larvae, with an average protein-to-lipid content ratio of 2.8. On average, 86% of protein synthesized was turned over (i.e. only 14% retained as protein accreted), with a regulatory shift in depositional efficiency resulting in increased protein accretion during later larval growth. Variation in protein depositional efficiency among families did not explain the range in larval growth rates. Instead, changes in protein synthesis rates predicted 72% of growth variation. High rates of protein synthesis to support faster growth, in turn, necessitated greater allocation of the total ATP pool to protein synthesis. An ATP allocation model is presented for larvae of C. gigas that includes the major components (82%) of energy demand: protein synthesis (45%), ion pump activity (20%), shell formation (14%) and protein degradation (3%). The metabolic trade-offs between faster growth and the need for higher ATP allocation to protein synthesis could be a major determinant of fitness for larvae of different genotypes responding to the stress of environmental change.

Experimental ocean acidification alters the allocation of metabolic energy.

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.

Increasing Temperature Results in Higher Allocation of Energy to Protein Synthesis in Sea Urchin Larvae (Lytechinus pictus).

AbstractIt is well established that metabolic processes change with temperature and size. Yet the underlying physiological mechanisms are less well understood regarding how such processes covary within a species and particularly so for developmental stages. Physiological analysis of larvae of the sea urchin Lytechinus pictus revealed that protein was the major biochemical substrate supporting metabolism. The complex dynamics of protein synthesis, turnover, and accretion changed during growth, showing a sevenfold decrease in the ratio of protein accretion to protein synthesis (protein depositional efficiency). To test hypotheses of physiological variation with rising temperature, larvae were reared over a temperature range experienced by this species in its ambient habitat. The thermal sensitivity of protein synthesis was greater than respiration (thermal sensitivity values of 3.7 and 2.4, respectively). Bioenergetic calculations revealed a disproportionate increase in energy allocation toward protein synthesis with rising temperature. These differential temperature sensitivities result in metabolic trade-offs of energy acquisition and expenditure, thereby altering physiological homeostasis. Such insights are of value for improving predictions about limits of biological resilience in a warming ocean.

Shifting Balance of Protein Synthesis and Degradation Sets a Threshold for Larval Growth Under Environmental Stress.

Exogenous environmental factors alter growth rates, yet information remains scant on the biochemical mechanisms and energy trade-offs that underlie variability in the growth of marine invertebrates. Here we study the biochemical bases for differential growth and energy utilization (as adenosine triphosphate [ATP] equivalents) during larval growth of the bivalve Crassostrea gigas exposed to increasing levels of experimental ocean acidification (control, middle, and high pCO2, corresponding to ∼400, ∼800, and ∼1100 µatm, respectively). Elevated pCO2 hindered larval ability to accrete both shell and whole-body protein content. This negative impact was not due to an inability to synthesize protein per se, because size-specific rates of protein synthesis were upregulated at both middle and high pCO2 treatments by as much as 45% relative to control pCO2. Rather, protein degradation rates increased with increasing pCO2. At control pCO2, 89% of cellular energy (ATP equivalents) utilization was accounted for by just 2 processes in larvae, with protein synthesis accounting for 66% and sodium-potassium transport accounting for 23%. The energetic demand necessitated by elevated protein synthesis rates could be accommodated either by reallocating available energy from within the existing ATP pool or by increasing the production of total ATP. The former strategy was observed at middle pCO2, while the latter strategy was observed at high pCO2. Increased pCO2 also altered sodium-potassium transport, but with minimal impact on rates of ATP utilization relative to the impact observed for protein synthesis. Quantifying the actual energy costs and trade-offs for maintaining physiological homeostasis in response to stress will help to reveal the mechanisms of resilience thresholds to environmental change.

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

Cold environments represent a substantial volume of the biosphere. To study developmental physiology in subzero seawater temperatures typically found in the Southern Ocean, rates and costs of protein synthesis were measured in embryos and larvae of Sterechinus neumayeri, the Antarctic sea urchin. Our analysis of the "cost of living" in extreme cold for this species shows (1) that cost of protein synthesis is strikingly low during development, at 0.41 +/- 0.05 J (mg protein synthesized)(-1) (n = 16); (2) that synthesis cost is fixed and independent of synthesis rate; and (3) that a low synthesis cost permits high rates of protein turnover at -1 degrees C, at rates comparable to those of temperate species of sea urchin embryos developing at 15 degrees C. With a low synthesis cost, even at the highest synthesis rates measured (gastrulae), the proportion of total metabolism accounted for by protein synthesis in the Antarctic sea urchin was 54%-a value similar to that of temperate sea urchin embryos. In the Antarctic sea urchin, up to 87% of metabolic rate can be accounted for by the combined energy costs of protein synthesis and the sodium pump. We conclude that, in Antarctic sea urchin embryos, high rates of protein synthesis can be supported in extreme-cold environments while still maintaining low rates of respiration.

Metabolic Cost of Protein Synthesis in Larvae of the Pacific Oyster (Crassostrea gigas) Is Fixed Across Genotype, Phenotype, and Environmental Temperature.

The energy made available through catabolism of specific biochemical reserves is constant using standard thermodynamic conversion equivalents (e.g., 24.0 J mg protein(-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 ± one standard error, n = 21) of 2.1 ± 0.2 J (mg protein synthesized)(-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.

Genetically Determined Variation in Developmental Physiology of Bivalve Larvae (Crassostrea gigas).

Understanding the complex interactions that regulate growth and form is a central question in developmental physiology. We used experimental crosses of pedigreed lines of the Pacific oyster, Crassostrea gigas, to investigate genetically determined variations in larval growth and nutrient transport. We show that (i) transport rates at 10 and 100 µM glycine scale differentially with size; (ii) size-specific maximum transport capacity (Jmax) is genetically determined; and (iii) Jmax serves as an early predictive index of subsequent growth rate. This relationship between genetically determined Jmax and growth suggests the potential use of transporter genes as biomarkers of growth potential. Analysis of the genome of C. gigas revealed 23 putative amino acid transporter genes. The complexity of gene families that underpin physiological traits has additional precedents in this species and others and warrants caution in the use of gene expression as a biomarker for physiological state. Direct in vivo measurements of physiological processes using species with defined genotypes are required to understand genetically determined variance of nutrient flux and other processes that regulate development and growth.

Energy metabolism during larval development of green and white abalone, Haliotis fulgens and H. sorenseni.

An understanding of the biochemical and physiological energetics of lecithotrophic development is useful for interpreting patterns of larval development, dispersal potential, and life-history evolution. This study investigated the metabolic rates and use of biochemical reserves in two species of abalone, Haliotis fulgens (the green abalone) and H. sorenseni (the white abalone). Larvae of H. fulgens utilized triacylglycerol as a primary source of endogenous energy reserves for development ( approximately 50% depletion from egg to metamorphic competence). Amounts of phospholipid remained constant, and protein dropped by about 30%. After embryogenesis, larvae of H. fulgens had oxygen consumption rates of 81.7 +/- 5.9 (SE) pmol larva(-1) h(-1) at 15 degrees C through subsequent development. The loss of biochemical reserves fully met the needs of metabolism, as measured by oxygen consumption. Larvae of H. sorenseni were examined during later larval development and were metabolically and biochemically similar to H. fulgens larvae at a comparable stage. Metabolic rates of both species were very similar to previous data for a congener, H. rufescens, suggesting that larval metabolism and energy utilization may be conserved among closely related species that also share similar developmental morphology and feeding modes.

Separating the nature and nurture of the allocation of energy in response to global change.

Understanding and predicting biological stability and change in the face of rapid anthropogenic modifications of ecosystems and geosystems are grand challenges facing environmental and life scientists. Physiologically, organisms withstand environmental stress through changes in biochemical regulation that maintain homeostasis, which necessarily demands tradeoffs in the use of metabolic energy. Evolutionarily, in response to environmentally forced energetic tradeoffs, populations adapt based on standing genetic variation in the ability of individual organisms to reallocate metabolic energy. Combined study of physiology and genetics, separating "Nature and Nurture," is, thus, the key to understanding the potential for evolutionary adaptation to future global change. To understand biological responses to global change, we need experimentally tractable model species that have the well-developed physiological, genetic, and genomic resources necessary for partitioning variance in the allocation of metabolic energy into its causal components. Model species allow for discovery and for experimental manipulation of relevant phenotypic contrasts and enable a systems-biology approach that integrates multiple levels of analyses to map genotypic-to-phenotypic variation. Here, we illustrate how combined physiological and genetic studies that focus on energy metabolism in developmental stages of a model marine organism contribute to an understanding of the potential to adapt to environmental change. This integrative research program provides insights that can be readily incorporated into individual-based ecological models of population persistence under global change.

Ribosomal analysis of rapid rates of protein synthesis in the Antarctic sea urchin Sterechinus neumayeri.

Previous research has shown that developing stages of the Antarctic sea urchin Sterechinus neumayeri have high rates of protein synthesis that are comparable to those of similar species living in much warmer waters. Direct measurements of the biosynthetic capacities of isolated ribosomes have not been reported for marine organisms living in the extreme-cold environment of Antarctica. Such measurements are required for a mechanistic understanding of how the critical and highly complex processes involved in protein synthesis are regulated in animals living in the coldest marine environment on Earth (< -1 degrees C). We tested the hypothesis that high rates of protein synthesis in the cold are a direct result of high biosynthetic capacities of ribosomes engaged in protein synthesis. Our results show that the rate at which ribosomes manufacture proteins (i.e., the peptide elongation rate) at -1 degrees C is surprisingly similar to rates measured in other sea urchin species at temperatures that are over 15 degrees C warmer. Average peptide elongation rates for a range of developmental stages of the Antarctic sea urchin were 0.36 codons s(-1) (+/- 0.05, SE). On the basis of subcellular rate determinations of ribosomal activity, we calculated stage-specific rates of protein synthesis for blastulae and gastrulae to be 3.7 and 6.5 ng protein h(-1), respectively. These findings support the conclusion that the high rates of biosynthesis previously reported for the Antarctic sea urchin are an outcome of high ribosomal activities.

Unequal and genotype-dependent expression of mitochondrial genes in larvae of the pacific oyster Crassostrea gigas.

Mitochondria are essential for regulation of energy metabolism, but little is known about patterns of mitochondrial genome expression in invertebrates. To explore the association of mitochondrial expression with differential growth of Crassostrea gigas, the Pacific oyster, we crossed two inbred lines to produce inbred and hybrid larvae, which grew at different rates under the same environmental conditions. Using high-throughput cloning and sequencing methods, we identified 1.1 million expressed sequence tags from the mitochondrial genome, 96.7% of which were perfect matches to genes targeted by the method. Expression varied significantly among genes, ranging over nearly four orders of magnitude, from mt:lRNA, which constituted 21% of all transcripts, to mt:CoII, which constituted less than 0.02% of all transcripts. Variable expression of genes coding for subunits of macromolecular complexes (e.g., mt:CoI and mt:CoII) implies that stoichiometry in these complexes must be regulated post-transcriptionally. Surprisingly, the mitochondrial transcriptome contained non-coding transcripts, which may play a role in the regulation of mitochondrial function. Finally, mitochondrial expression depended strongly on maternal factors and nuclear-cytoplasmic interactions, which may explain previously observed growth differences between reciprocal hybrids. Differences in mitochondrial gene expression could provide a biochemical index for the metabolic basis of genetically determined differences in larval growth.

Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus).

Transport of amino acids from low concentrations in seawater by marine invertebrates has been extensively studied, but few of the genes involved in this physiological process have been identified. We have characterized three amino acid transporter genes cloned from embryos of the sea urchin Strongylocentrotus purpuratus. These genes show phylogenetic proximity to classical amino acid transport systems, including Gly and B0+, and the inebriated gene (INE). Heterologous expression of these genes in frog oocytes induced a 40-fold increase in alanine transport above endogenous levels, demonstrating that these genes mediate alanine transport. Antibodies specific to one of these genes (Sp-AT1) inhibited alanine transport, confirming the physiological activity of this gene in larvae. Whole-mount antibody staining of larvae revealed expression of Sp-AT1 in the ectodermal tissues associated with amino acid transport, as independently demonstrated by autoradiographic localization of radioactive alanine. Maximum rates of alanine transport increased 6-fold during early development, from embryonic to larval stages. Analysis of gene expression during this developmental period revealed that Sp-AT1 transcript abundance remained nearly constant, while that of another transporter gene (Sp-AT2) increased 11-fold. The functional characterization of these genes establishes a molecular biological basis for amino acid transport by developmental stages of marine invertebrates.

Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas).

Compared with understanding of biological shape and form, knowledge is sparse regarding what regulates growth and body size of a species. For example, the genetic and physiological causes of heterosis (hybrid vigor) have remained elusive for nearly a century. Here, we investigate gene-expression patterns underlying growth heterosis in the Pacific oyster (Crassostrea gigas) in two partially inbred (f = 0.375) and two hybrid larval populations produced by a reciprocal cross between the two inbred families. We cloned cDNA and generated 4.5 M sequence tags with massively parallel signature sequencing. The sequences contain 23,274 distinct signatures that are expressed at statistically nonzero levels and show a highly positively skewed distribution with median and modal counts of 9.25 million and 3 transcripts per million, respectively. For nearly half of these signatures, expression level depends on genotype and is predominantly nonadditive (hybrids deviate from the inbred average). Statistical contrasts suggest approximately 350 candidate genes for growth heterosis that exhibit concordant nonadditive expression in reciprocal hybrids; this represents only approximately 1.5% of the >20,000 transcripts. Patterns of gene expression, which include dominance for low expression and even underdominance of expression, are more complex than predicted from classical dominant or overdominant explanations of heterosis. Preliminary identification of ribosomal proteins among candidate genes supports the suggestion from previous studies that efficiency of protein metabolism plays a role in growth heterosis.

Fixed metabolic costs for highly variable rates of protein synthesis in sea urchin embryos and larvae.

Defining the physiological mechanisms that set metabolic rates and the 'cost of living' is important for understanding the energy costs of development. Embryos and larvae of the sea urchin Lytechinus pictus (Verrill) were used to test hypotheses regarding differential costs of protein synthesis in animals differing in size, rates of protein synthesis, and physiological feeding states. For embryos, the rate of protein synthesis was 0.22+/-0.014 ng protein embryo(-1) h(-1) (mean +/- s.e.m.) and decreased in unfed larvae to an average rate of 0.05+/-0.001 ng protein larva(-1) h(-1). Fed larvae had rates of synthesis that were up to 194 times faster than unfed larvae (9.7+/-0.81 ng protein larva(-1) h(-1)). There was no significant difference, however, in the cost of protein synthesis between these larvae with very different physiological states. Furthermore, the cost of synthesis in the larval stages was also similar to costs measured for blastula and gastrula embryos of 8.4+/-0.99 J mg(-1) protein synthesized. The cost of protein synthesis was obtained using both direct ('inhibitor') and indirect ('correlative') measurements; both methods gave essentially identical results. Protein synthesis accounted for up to 54+/-8% of metabolic rate in embryos. Percent of metabolism accounted for by protein synthesis in larvae was dependent on their physiological feeding state, with protein synthesis accounting for 16+/-4% in unfed larvae and 75+/-11% in fed larvae. This regulation of metabolic rate was due to differential rates of synthesis for a fixed energy cost per unit mass of protein synthesized. The cost of synthesizing a unit of protein did not change with increasing rates of protein synthesis. We conclude that the cost of protein synthesis is independent of the rate of synthesis, developmental stage, size and physiological feeding state during sea urchin development.

Amino Acid Uptake and Metabolism by Larvae of the Marine Worm Urechis caupo (Echiura), a New Species in Axenic Culture.

Axenic (bacteria-free) larval cultures of the marine echiuran worm, Urechis caupo, were reliably obtained by aseptically removing gametes directly from the gamete storage organs. Trochophore larvae only removed neutral amino acids from seawater as measured by high-performance liquid chromatography (HPLC). There was no detectable uptake, as measured by HPLC, of acidic or basic amino acids. Kinetic analysis showed that the transport system for alanine in 4-day-old larvae had a Kt of 4-6 µM and a Jmax of 9-10 pmol larva-1 h-1. Following a 50-min exposure, the majority of the radioactivity (95%) from 14C-alanine was found in the trichloroacetic acid-soluble fraction. Very little label appeared as acid-insoluble material, and there was no detectable lipid biosynthesis from 14C-alanine. Approximately 12% of the total alanine transported was released in the form of 14CO2. Thin-layer chromatography of intracellular free amino acid pools demonstrated that aspartic acid and glutamic acid were radiolabeled from the alanine precursor. A comparison of the energy acquired from the transport of alanine, with the metabolic rate of 4-day-old larvae, revealed that 51% of the metabolic demand could be provided by the transport and complete catabolism of this single amino acid at a concentration of 595 nM in seawater.