Lytechinus Pictus Research Articles

Ocean acidification alters skeletogenesis and gene expression in larval sea urchins

Ocean acidification, the reduction of ocean pH due to the absorption of anthropogenic atmospheric CO2, is expected to influence marine ecosystems through effects on marine calcifying organisms. These effects are not well understood at the community and ecosystem levels, although the consequences are likely to involve range shifts and population declines. A current focus in ocean acidification research is to understand the resilience that organisms possess to withstand such changes, and to extend these investigations beyond calcification, addressing impacts on other vul- nerable physiological processes. Using morphometric methods and gene expression profiling with a DNA microarray, we explore the effects of elevated CO2 conditions on Lytechinus pictus echino- plutei, which form a calcium carbonate endoskeleton during pelagic development. Larvae were raised from fertilization to pluteus stage in seawater with elevated CO2. Morphometric analysis showed significant effects of enhanced CO2 on both size and shape of larvae; those grown in a high CO2 environment were smaller and had a more triangular body than those raised in normal CO2 con- ditions. Gene expression profiling showed that genes central to energy metabolism and biomineral- ization were down-regulated in the larvae in response to elevated CO2, whereas only a few genes involved in ion regulation and acid-base balance pathways were up-regulated. Taken together, these results suggest that, although larvae are able to form an endoskeleton, development at elevated CO2 levels has consequences for larval physiology as shown by changes in the larval transcriptome.

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca(2+) release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.

In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry

Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.

Culture of benthic diatoms and effect of dissolved organic carbon on metamorphosis of the ‘white sea urchinLytechinus pictus(Verrill)’

The effect of biofilms of the benthic diatom Navicula incerta and of the dissolved organic carbon (DOC) they produce was evaluated on the growth and survival of Lytechinus pictus postlarvae during metamorphosis. In each container, 300 postlarvae were exposed to biofilms beginning on days 1, 3 and 6 of metamorphosis, using three replicates and a blank containing only filtered water. The experiments were run for 15 and 18 days in 500-mL containers with daily water changes. We measured 30 postlarvae from each container and counted all organisms on days 1, 3, 9 and 15; DOC was quantified on days 3, 6 and 9. A second experiment was conducted to evaluate whether the presence of diatoms or the DOC they produced improved postlarval development. Five assays were performed in triplicate: a positive control with biofilms, a negative control with filtered water and three treatments with DOC-enriched water from a N. incerta culture supplied on days 1, 4 and 8. In both experiments, postlarvae attained the largest size and highest survival in the treatments with the biofilm. The results indicate that the increase in postlarval size and survival was due to the DOC produced by the diatoms and not by their presence alone.

Sniffing Out New Data and Hypotheses on the Form, Function, and Evolution of the Echinopluteus Post-Oral Vibratile Lobe

The performance requirements of ciliary band feeding explain the convoluted forms of many marine invertebrate larvae. Convolutions increase surface area and therefore feeding rates per unit body volume. We review recent advances in morphology, neural development, and behavior at settlement of the echinoid Lytechinus pictus and provide new ultrastructural and expression data on larvae of its congener, L. variegatus. Larvae of the echinometrid Colobocentrotus atratus contain neurons identified by their expression of nitric oxide synthase (NOS), indicating that this character is not unique to Lytechinus. We hypothesize that in some echinoids the convoluted shape of the post-oral vibratile lobe (POVL) covaries with the distribution of identified sensory neurons to enable olfaction during settlement. An analysis of variation in structural elaboration of the post-oral transverse ciliary band (PTB) within Echinoida and in feeding larvae of other echinoderm classes indicates that only echinoids, but not all echinoids, possess this novel character; larvae that do are distributed heterogeneously within the class. In recognition of this specialized function for the POVL and surrounding ectoderm, and because it is lobate and grows toward the mouth, we propose naming this structure the adoral lobe.

Periodate oxidation of sea urchin hyalin(s): A glycobiological study towards understanding of hyalin's function as a specific cell adhesion molecule

Hyalin (Hy), a set of large glycoproteins containing 2‐3% carbohydrate appears to be a specific cell adhesion molecule in the sea urchin embryo model. Sodium (meta) periodate (PI) can selectively degrade carbohydrate by oxidizing the C‐C bonds between vicinal diols. Untreated Strongylocentrotus purpuratus Hy blocked the adhesion of the tip of the archenteron to the roof of the blastocoel (the specific adhesive interaction under study) in two assays: (1) a microplate assay using live Lytechinus pictus embryos and (2) using dissected pieces of the archenteron and blastocoel roof. Hy cross reacts between these two species. PI treated Hy did not block this interaction in either assay. The microplate analysis, with over 16,000 L. pictus embryos, indicated that 6 hr PI treated Hy (0.17‐0.34 mg treated Hy/ml low calcium artificial sea water ‐LCSW) did not block the cellular interaction, while untreated Hy (0.17‐0.34 mg untreated Hy/ml LCSW) did. 76.4‐77.2% ± 2% of the embryos in PI treated Hy possessed complete archenterons, while 2.7‐7.6% ± 12% possessed complete archenterons in the untreated Hy samples with a p value of less than 0.001. Similar results were obtained using the dissected pieces assay. While this study suggests that Hy carbohydrate is involved in Hy function, further studies are needed to determine if PI is also affecting Hy in other ways (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).

A novel approach for investigating glycan involvement in embryonic cellular interactions. II. lectin studies

Here we investigate the effects of 5 lectins at concentrations ranging from 0.0005 mg lectin ‐ 0.5 mg lectin per ml low calcium sea water on specific cellular interactions in the NIH model sea urchin embryo Lytechinus pictus using a microplate assay. 3 separate experiments were conducted with 12 microwells in each experiment at each lectin concentration, yielding a total of 36 wells with hundreds of embryos assessed for each lectin concentration. Lectins(or no lectin controls) were added at 24 hrs of development and the effects on archenteron/elongation/binding to the blastocoel roof were recorded after an additional 24 hrs. Phytolacca americana (specific for N‐acetyl‐beta‐D‐glucosamine residues) inhibited archenteron elongation/attachment to the blastocoel roof in a concentration dependent manner when controls and treated embryos were compared (p less than 0.0001 comparing complete archenterons in controls versus experimentals). Pisum sativum (specific for alpha‐D‐glucose and alpha‐D‐mannose) and Ulex europaeus UEA 1 (specific for L‐fucose) lectins both caused exogastrulation, where not only does the archenteron not attach but it everts from the embryo proper (p value of less than 0.0001 comparing exogastrulation in controls versus experimentals). Helix pomata (specific for N‐acetyl‐alpha‐D‐galactosamine) and Glycine max (specific for N‐acetyl‐alpha‐D‐galactosamine and D‐galactose) had no effects on the embryos, like the controls.The results indicate that specific lectins that bind to specific saccharides influence a model cellular interaction. Most important, however, is that the microplate approach used here is able to quantitatively assess specific probed events in living embryos (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC , the Joseph Drown Foundation and the Sidney Stern Memorial Trust).

A novel approach for investigating glycan involvement in embryonic cellular interactions. III. whole embryo periodate studies

Periodate oxidation has been widely used to degrade the carbohydrate components of molecules to help determine the role of sugars in molecular function. In this study, 24.5‐26 hr early gastrula Lytechinus pictus sea urchin embryos were incubated with specific ultra‐low concentrations of sodium (meta) periodate that degrades some carbohydrates by selectively oxidizing C‐C bonds between vicinal diols. Incubations were carried out in 15 degrees C artificial seawater in 96 well microplates for 24‐25 hrs. After the incubation period, embryos were examined for viability, fixed in 5% formaldehyde, and the morphological characteristics of 5664 embryos were quantitatively assessed as embryos possessing complete attached archenterons, incomplete unattached archenterons, no invagination, exogastrulation, or embryos that were dead. At 1.2x10−6 ‐3.9x10−5 mg periodate/ml 15‐30% of the embryos were swimming and possessed incomplete unattached archenterons, while the controls without periodate displayed 2.3% incomplete archenterons. At higher concentrations of periodate (7.8x10−5‐1.0x10−2 mg periodate/ml), embryo death occurred. Using a two‐sample equal variances t‐test, the numbers of unattached archenterons in the 1.2x10−6‐3.9 x10−5 mg periodate/ml treated embryos were significantly different from those in the controls without periodate at a p value of less than 0.001. The use of periodate in probing whole, living embryos is novel and could become widely used to help identify the role of glycans in interacting systems (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation, and the Sidney Stern Memorial Trust).

Role of LSM34/SpSM50 proteins in endoskeletal spicule formation in sea urchin embryos

Abstract. Sea urchin embryos form an endoskeletal spicule composed of calcium carbonate and occluded matrix proteins. The accumulation of the LSM34 spicule matrix protein in embryos of Lytechinus pictus (and its ortholog, SpSM50, in Strongylocentrotus purpuratus) has been inhibited using morpholino antisense oligonucleotides. The inhibition, using relatively high levels of antisense reagent, can result in the complete absence of spicules, and the complete loss of immunoreactive LSM34/SpSM50, as judged by immunostaining and Western blotting. Primary mesenchyme cells (PMCs) do form and express PMC‐specific cell surface antigens despite this inhibition. However, these anti‐LSM34/SpSM50‐treated embryos do not accumulate SM30 protein, another major matrix protein. Hence, both the initiation of spicule formation and subsequent morphogenesis require LSM34 accumulation in L. pictus, and the accumulation of its ortholog, SpSM50, in S. purpuratus.

Serotonin synthesis is necessary for gastrulation in the sea urchin, Lytechinus pictus

Serotonin synthesis is necessary for gastrulation in the sea urchin, Lytechinus pictus

Bipolar, anastral spindle development in artificially activated sea urchin eggs.

The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized by means of the centriole of the fertilizing sperm. In this study, we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5, and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral; displayed metaphase and anaphase-like stages; labeled for centrosomal material, NuMA, and the three mitotic kinesins; and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 microg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin-hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.

Bisphenol A directly targets tubulin to disrupt spindle organization in embryonic and somatic cells.

There is increasing concern that animal and human reproduction may be adversely affected by exposure to xenoestrogens that activate estrogen receptors. There is evidence that one such compound, Bisphenol A (BPA), also induces meiotic and mitotic aneuploidy, suggesting that these kinds of molecules may also have effects on cell division. In an effort to understand how Bisphenol A might disrupt cell division, a phenotypic analysis was carried out using sea urchin eggs, whose early embryonic divisions are independent of zygotic transcription. Fertilized Lytechinus pictus eggs exposed to BPA formed multipolar spindles resulting in failed cytokinesis in a dose-dependent, transcriptionally independent manner. By use of novel biotinylated BPA affinity probes to fractionate cell-free extracts, tubulin was identified as a candidate binding protein by mass spectrometry, and BPA promoted microtubule polymerization and centrosome-based microtubule nucleation in vitro but did not appear to display microtubule-stabilizing activity. Treatment of mammalian cells demonstrated that BPA as well as a series of Bisphenol A derivatives induced ectopic spindle pole formation in the absence of centrosome overduplication. Together, these results suggest a novel mechanism by which Bisphenol A affects the nucleation of microtubules, disrupting the tight spatial control associated with normal chromosome segregation, resulting in aneuploidy.

Development of nitric oxide synthase‐defined neurons in the sea urchin larval ciliary band and evidence for a chemosensory function during metamorphosis

We previously reported that initiation of metamorphosis of larvae of Lytechinus pictus is negatively regulated by nitric oxide (NO) and cGMP. We have examined the expression of nitric oxide synthase (NOS) and cGMP in cells of the developing larva. A section of the post-oral ciliary band of feeding larvae includes neural cells defined by their expression of both NOS and the echinoderm neural-specific antibody 1E11. These neurons project processes to the pre-oral neuropile during larval development. Larvae regenerated this section of the ciliary band after its excision, complete with NOS-defined neurons that projected again to the pre-oral neuropile. Excision of ectoderm containing the post-oral ciliary band prevented a behavioral and morphogenetic response of competent larvae to biofilm, and delayed initiation of metamorphosis. Elevated cGMP levels were detected in several larval and juvenile cell types prior to metamorphosis. Treatment of larvae with ODQ, an inhibitor of soluble guanylate cyclase, decreased cGMP levels and induced metamorphosis while a generator of NO counteracted this effect, indicating inhibition of metamorphosis by NO operates via interaction with soluble guanylate cyclase. We discuss these observations, proposing that the NOS-defined neurons in the post-oral ciliary band have a chemosensory function during settlement and metamorphosis that involves morphologically specialized ectoderm and manipulation of fluid flow. We provide a tentative cellular model of how environmental signals may be transduced into a metamorphic response.

Interspecies Effects of Sea Urchin Hyalin on Archenteron Elongation and Attachment

The sea urchin embryo is a simple model system to study the molecular basis of cellular interactions that may be important in less accessible higher organisms. Recently, using a microassay to quantitatively evaluate the effects of exogenously added molecules on sea urchin gastrulation, we found that hyalin, a large sea urchin glycoprotein purified from the hyaline layer, dramatically affected gastrulation (Razinia, et al., Zygote, in press; see abstract 307 in these proceedings). In this study, hyalin isolated from Strongylocentrotus purpuratus embryos was tested for its effects on another species of sea urchin, Lytechinus pictus. Fifty seven (57) micrograms of purified S. purpuratus hyalin/ml, in low calcium artificial sea water (ASW), were incubated with 24 hr L. pictus embryos in 96-well polystyrene flat-bottom microplates, containing about 12 embryos per well, for 24 hrs. Control wells contained ASW, NaCl-KCl low calcium ASW or low calcium ASW. The results from replicate experiments were plotted as mean percentages of embryos exhibiting the following archenteron morphologies: complete attached archenterons, unattached archenterons, no invagination, exogastrulated archenterons, plus/minus standard error of the mean. Most of the hyalin treated embryos exhibited unattached archenterons, while in the controls, the archenterons were complete and attached to the blastocoel roof. Hyalin, therefore, has interspecies effects and may be involved in archenteron elongation and/or attachment to the blastocoel roof (Supported by NIH NIGMS SCORE, RISE,MARC and the Joseph Drown Foundation).

Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis

Metamorphosis in marine invertebrate larvae is a dynamic, environmentally dependent process that integrates ontogeny with habitat selection. The capacity of many marine invertebrate larvae to survive and maintain metamorphic competence in the absence of environmental cues has been hypothesized to be an adaptive convergence (Hadfield and others 2001). A survey of the literature reveals that a single generalized hypothesis about metamorphic competence as an adaptive convergence is not sufficient to account for interspecific variation in this character. In an attempt to capture this variation, we discuss the "desperate larva hypothesis" and propose two additional hypotheses called the "variable retention hypothesis" and the "death before dishonor hypothesis." To validate these additional hypotheses we collected data on taxa from the published literature and performed a contingency analysis to detect correlations between spontaneous metamorphosis, habitat specificity and/or larval life-history mode, three characters relevant to environmentally induced settlement and metamorphosis. In order to account for phylogenetic bias in these correlations, we also constructed a phylogeny of these taxa and again performed a character-correlation analysis. Both these tests suggest that juvenile habitat specificity is correlated to the capacity of individuals to retain the competent larval state in the absence of substrate cues and therefore validate the existence of more than one hypothesis about metamorphic competence. We provide new data from the sea urchin Lytechinus pictus that suggest that nitric oxide (NO) and thyroxine hormone signaling interact to determine the probability of settlement in response to a settlement cue. Similarly, we provide evidence that thyroxine signaling in the sand dollar Dendraster excentricus increases spontaneous metamorphosis in the absence of cues from adult conspecifics in a manner that is independent of larval age.

A novel model for studying adhesive interactions

A classic model cellular interaction is the adhesion of the tip of the advancing archenteron to the blastocoel roof during gastrulation in the sea urchin embryo. Here we present a novel approach to study cellular interactions, using this specific model, in pristine environments, away from all confounding factors. Thirty-one fixed (3.7% formaldehyde) and 39 live 48-54 hour Lytechinus pictus gastrula embryos were dissected using insect pins to obtain isolated pieces of the blastocoel roof and archenteron. These pieces adhered to each other as they do in the whole embryo. Fixed pieces were preferable to live because they did not lose structural integrity or move. Fixed pieces from 54 hour embryos bound to FITC-Lens culinaris agglutinin (0.045 micrograms per microliter) and this binding was inhibited by 0.2 M alpha-methyl-mannose. Lens culinaris agglutinin reduced the adhesiveness of the pieces with each other, suggesting that the ligands to which the lectin binds may be involved in the adhesion of archenteron and blastocoel roof, as also suggested in whole live embryos (Acta Histochemica 100: 193–200, 1998; 101: 293–303, 1999). These results were obtained in 3 repeated experiments with 16 embryos. Study of isolated pieces of cellular interactions is a novel approach for investigating adhesive mechanisms in pristine environments. (supported by NIH NIGMS SCORE, RISE, ITQ program and the Joseph Drown Foundation).

Microplate assay for quantitative analysis of developmental effects

It is often difficult to determine the activity of physiologically functional molecules during purification procedures because of the lack of appropriate quantitative microassays. The sea urchin embryo is a NIH designated model system that can provide insights into physiological mechanisms in higher organisms. Here, a microplate assay has been developed for quantitatively evaluating the effects of various molecular probes such as cell adhesion molecules, disaggregation supernatants, HPLC fractions and other preparations on living sea urchin embryos. Using wide-mouthed pipette tips, 17 or 25 microliters of specific stage Lytechinus pictus embryos were transferred in sea water to each well of a 96 well microplate, at an embryo concentration that resulted in about 10 embryos per well. Defined volumes of solutions to be tested and control samples of sea water only were added to the wells with the embryos, incubated for specific times at 15 C, and the embryos were observed live and after fixation with 5 microliters of 10% formaldehyde using an inverted light microscope at 100X–200X magnification. The small number of embryos in each well made evaluation of the effects of the added material that was tested easy. Specific embryo characteristics, such as numbers of exogastrulated embryos, were easily scored and the results in experimental versus control samples were statistically analyzed. We are using this assay during the purification of putative sea urchin cell adhesion molecules. The complete transparency and easy availability of sea urchin embryos make them ideal for studying molecular mechanisms that may be similar in higher organisms (supported by NIH NIGMS SCORE, RISE, MARC, ITQ program and the Joseph Drown Foundation).

Calcium-responsive contractility during fertilization in sea urchin eggs.

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle.

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.