Anaerobic dormancy quantified in artemia embryos: a calorimetric test of the control mechanism.

Continuous measurement of heat dissipation from brine shrimp embryos during reversible transitions from aerobic development to anaerobic dormancy demonstrates a primary role for intracellular pH(pH(i))in this metabolic switching. Artificially elevating the depressed pH(i) during anoxia by adding ammonia markedly reactivates metabolism, as judged by increases in heat dissipation, trehalose catabolism, and the ratio of adenosine triphosphate to adenosine diphosphate. Energy flow during anaerobic dormancy is suppressed to 2.4 percent of aerobic values, which is the lowest percentage thus far reported for euryoxic animals. Use of diguanosine tetraphosphate stores cannot account for this observed heat dissipation. Thus, mobilizing trace amounts of trehalose may explain the energy metabolism during quiescence.

pH-Induced Metabolic Transitions in Artemia Embryos Mediated by a Novel Hysteretic Trehalase.

Gastrula-stage embryos of the brine shrimp Artemia undergo reversible transitions between metabolically active and dormant states that are promoted by changes in intracellular pH. A macromolecular mechanism for this suppression of energy metabolism that involves regulation of the enzyme trehalase is reported here. Isolated trehalase from these embryos existed in two active forms that interconverted when exposed to physiological transitions in pH. This hysteretic interconversion was reversible, required minutes for completion, and involved a change in enzyme polymerization. The two states differed twofold in molecular size and were distinguishable electrophoretically. Compared to the smaller species, the polymerized form was strongly inhibited by acidic pH, adenosine 5'-triphosphate, and the substrate trehalose. Thus, the shift in assembly equilibrium toward the aggregated enzyme caused by pH values less than or equal to 7.4 may mediate the arrest of trehalose-fueled metabolism and respiration during dormancy in this cryptobiotic organism.

Living with water stress: evolution of osmolyte systems.

Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and combinations of urea and methylamines are the three types of osmolyte systems found in all water-stressed organisms except the halobacteria. The selective advantages of the organic osmolyte systems are, first, a compatibility with macromolecular structure and function at high or variable (or both) osmolyte concentrations, and, second, greatly reduced needs for modifying proteins to function in concentrated intracellular solutions. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing or favorable effects of osmolytes on macromolecular-solvent interactions.

Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus.

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via (14)CO(2)) into perchloric acid-precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole-embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.

Reversible depression of oxygen consumption in isolated liver mitochondria during hibernation.

The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25 degrees C. Relative to mitochondria from summer-active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP-dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.

Heat dissipation during long-term anoxia in Artemia franciscana embryos: identification and fate of metabolic fuels.

Microcalorimetric measurements of brine shrimp embryos during 6 days of anoxia indicated that heat dissipation was rapidly suppressed to 2.7% of control (aerobic) values over the first 9 h. Energy flow continued to decline slowly to 31 microW.g dry mass-1 (0.4% of control) during the subsequent 5.5 days. Within 2 h after returning anoxic embryos to aerobic conditions, heat dissipation rose to 77% of control rates. The calorimetric/respirometric (CR) ratio across this 2-h recovery period increased steadily from -226 to -346 kJ.mol O2-1). Prior to the anoxic exposures, hydrated embryos were incubated aerobically for 10 h to insure full initiation of carbohydrate metabolism (CR ratio = -484 kJ.mol O2-1). During the 6-day asymptotic approach to a nearly ametabolic state, trehalose and glycogen levels declined 18% and 13%, respectively. The majority of this utilization occurred within the first three days. Thermochemical calculations showed that carbohydrate catabolism accounted for 84% of the total heat dissipation measured over the 6-day anoxic bout; only 3% of the heat could be explained by the catabolism of diguanosine tetraphosphate (Gp4G). Analyses of embryo extracts by high performance liquid chromatography indicated that multiple acid end products were accumulated. Lactate and propionate reached 4.5 mM and 1.0 mM, respectively, but these compounds did not account quantitatively for the amount of carbohydrate utilized. However, the largest chromatographic peak that accumulated under anoxia has not been successfully identified. Fumarate and pyruvate levels decreased as anoxia proceeded. Thus, a perceptible energy flow in Artemia franciscana embryos still remained after 6 days of anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Quiescence in Artemia franciscana embryos: reversible arrest of metabolism and gene expression at low oxygen levels.

Depression of the production and consumption of cellular energy appears to be a prerequisite for the survival of prolonged bouts of anoxia. A correlation exists between the degree of metabolic depression under anoxia and the duration of anoxia tolerance. In the case of brine shrimp (Artemia franciscana) embryos, oxygen deprivation induces a reversible quiescent state that can be tolerated for several years with substantial survivorship. A global arrest of cytoplasmic translation accompanies the transition into anoxia, and rates of protein synthesis in mitochondria from these embryos appears to be markedly reduced in response to anoxia. Previous evidence suggests that the acute acidification of intracellular pH (pHi) by over 1.0 unit during the transition into anoxia contributes to the depression of biosynthesis, but message limitation does not appear to play a role in the down-regulation in either cellular compartment. The ontogenetic increase in mRNA levels for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin is blocked by anoxia and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). Further, the levels of COX I and actin mRNA do not decline appreciably during 6 h bouts of quiescence, even though protein synthesis is acutely arrested across this same period. Thus, the constancy of mRNA levels during quiescence indicates that reduced protein synthesis is not caused by message limitation but, instead, is probably controlled at the translational level. This apparent stabilization of mRNA under anoxia is mirrored in an extension of protein half-life. The ubiquitin-dependent pathway for protein degradation is depressed under anoxia and aerobic acidosis, as judged by the acute drop in levels of ubiquitin-conjugated proteins. Mitochondrial protein synthesis is responsive to both acidification of pHi and removal of oxygen per se. Matrix pH declines in parallel with pHi, and evidence from experiments with nigericin indicates that mitochondrial protein synthesis is depressed directly by acidification of matrix pH. The oxygen dependency of organellar protein synthesis is not explained by blockage of the electron transport chain or by the increased redox state. Rather, this cyanide- and antimycin-insensitive, but hypoxia-sensitive, inhibitory signature for the arrest of protein synthesis suggests the presence of a molecular oxygen sensor within the mitochondrion.

Oxygen, pHi and arrest of biosynthesis in brine shrimp embryos.

Embryos of the brine shrimp Artemia franciscana are able to withstand bouts of environmental anoxia for several years by entering a quiescent state, during which time metabolism is greatly depressed. Within minutes of oxygen removal, intracellular pH (pHi) drops at least 1.0 unit. This acidification has been strongly implicated in the arrest of both catabolic and anabolic processes in the cytoplasm. A global arrest of cytoplasmic translation accompanies the transition into anoxia or into aerobic acidosis (artificial quiescence imposed by intracellular acidification with CO2 in the presence of oxygen). Similarly, protein synthesis in isolated mitochondria from these embryos is also reduced markedly in response to acidic pH (80% reduction) or anoxia (79% reduction). The constancy of mRNA levels during quiescence indicates that protein synthesis is likely to be controlled at the translational level. Mitochondrial matrix pH is 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient is abolished with the K+/H+ ionophore nigericin, the extramitochondrial pH optimum for protein synthesis displays an alkaline shift of approximately 0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion. The oxygen dependency of mitochondrial protein synthesis is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depresses protein synthesis by 77% after 1 h, normoxic incubations with saturating concentrations of cyanide or antimycin A have only a modest effect (36% reduction, cyanide; 20%, antimycin A). This cyanide- and antimycin-insensitive, but hypoxia-sensitive, inhibitory signature for the arrest of protein synthesis suggests the presence of a molecular oxygen sensor within the mitochondrion.

Arrest of cytochrome-c oxidase synthesis coordinated with catabolic arrest in dormant Artemia embryos.

We have examined cytochrome-c oxidase (COX) biosynthesis in brine shrimp (Artemia franciscana) embryos during preemergence development (PED), as well as its inhibition under anaerobic dormancy, to determine whether transitions in intracellular pH (pHi) have a regulatory influence on anabolic processes. Under control aerobic conditions (embryo pHi greater than or equal to 7.9), incorporation of radiolabeled amino acids shows that substantial biosynthesis of COX occurs during 12 h of PED (500% increase when corrected for enzyme turnover). This anabolic process is blocked under anoxia, a condition known to foster intracellular acidification (pHi less than or equal to 6.8). The arrest of COX synthesis is quantitatively identical when embryos are incubated aerobically during artifical acidification with CO2 (pHi = 6.8). The data suggest that pHi, directly or indirectly, is a regulator of protein synthesis in Artemia embryos during anaerobic dormancy. Previous work has established a fundamental role for pHi in the arrest of carbohydrate catabolism under anoxia. Thus there appears to be a coordinated suppression of energy-producing and energy-utilizing events as Artemia embryos enter quiescence that involves pHi as the common intracellular signal.

Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate.

Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics.

pH-induced hysteretic properties of phosphofructokinase purified from rat myocardium.

Phosphofructokinase (PFK) purified from the rat myocardium is reversibly inactivated under a pH regime approximating that reported for ischemic hearts. At pH 6.5 and 37 degrees C, the enzyme displays a hysteretic loss of activity during 60-min incubations, declining to 48% of control (pH 7.1, 37 degrees C) values. Citric acid increases the degree of inactivation (28% of control), whereas fructose 1,6-bisphosphate reduces the decline in activity. Simultaneous measurements of 90 decreases light scattering and catalytic activity suggest the inactivation is temporally linked to dissociation of active tetrameric enzyme into an inactive form of lower molecular weight. Fluorescence enhancement of the extrinsic probe sodium mansate, which binds preferentially to dimeric PFK, indicates that the equilibrium dimer concentration (cp1 infinity) increases as pH is lowered. This increase in cp1 infinity exhibits a strong inverse correlation (r = 0.984) with catalytic activity across the pH range of 8.0 to 6.5. Returning solution pH to 7.0 or above promotes a time-dependent reactivation and repolymerization of PFK. The rate of reactivation is increased at higher enzyme concentrations and in the presence of trimethylamine-N-oxide, a nitrogenous osmolyte noted for its ability to promote protein aggregation reactions. Thus these results demonstrate the capacity of rat heart PFK to undergo reversible inactivation and dissociation in vitro and represent the first phase of a two-part study testing the hypothesis that these pH-induced hysteretic processes are operative in the ischemic myocardium. The data are evaluated in terms of the potential roles of hysteretic enzymes in metabolic regulation.

Reversible dissociation and inactivation of phosphofructokinase in the ischemic rat heart.

To determine if shifts in the ratio of active phosphofructokinase (PFK) tetramers to the inactive dimeric form of the enzyme occur in vivo in the ischemic rat heart, we have developed a rocket immunoelectrophoretic (IEP) assay that provides a sensitive means by which to measure relative differences in this ratio among crude heart extracts. In ischemic hearts, in the face of a drop in intracellular pH from 6.95 to 6.25, there is a time-dependent decrease (63%) in the ratio of tetramer to dimer IEP rocket height relative to perfused controls. Concomitant with this hysteretic depolymerization is a 50% loss of PFK catalytic activity. Realkalinizing extracts of ischemic hearts fosters a recovery of 86% of the activity lost during ischemia and a return of the tetramer-to-dinner ratio to near control value. The amount of reactivation is directly dependent on the degree of enzyme dissociation that occurred during ischemia. Importantly, ischemia-induced dimerization is also reversed in vivo by postischemic reperfusion. The data are consistent with those in the previous study [Hand and Carpenter, Am. J. Physiol. 250 (Regulatory Integrative Comp. Physiol. 19): R505-R511, 1986] that characterized the pH-dependent hysteretic dissociation of heart PFK in vitro, and together they represent the first demonstration that this molecular behavior is operative in intact tissue. Other vertebrate muscle systems in which this mechanism might be functioning during pH-dependent glycolytic inhibition are discussed.

Regulatory features of protein synthesis in isolated mitochondria from Artemia embryos.

Optimal conditions were developed for measuring rates of protein synthesis in isolated mitochondria from encysted embryos of Artemia franciscana to 1) identify the required chemical constituents, 2) assess the influence of extramitochondrial pH on protein synthesis, and 3) investigate potential mechanisms coordinating nuclear and mitochondrial gene expression. Isolation procedures resulted in intact, highly coupled mitochondria [respiratory control ratio = 6.48 +/- 0.43 (SE), n = 21]. Requirements for maximal rates of protein synthesis, measured as incorporation of [3H]leucine (60 microM), included an oxidizable carbon source (10 mM succinate), adenine nucleotides (1.5 mM ADP), phosphate (10 mM), K+ (125 mM), Mg2+ (10 mM), amino acids (0.3 mM of each), sucrose or trehalose (500 mM), EGTA (1 mM), and bovine serum albumin (1 mg/ml). Rates were linear for 60 min at 25 degrees C (r = 0.99). Fluorography of translated products revealed 13 peptides. Previous research has shown that anoxia-induced acidification of intracellular pH (pHi) results in suppression of protein biosynthesis, as judged by cytochrome-c oxidase synthesis. In the present study, mitochondrial protein synthesis was acutely sensitive to external pH, with 80% inhibition observed by lowering pH from 7.5 to 6.8. Thus acidification of pHi may serve as one intracellular signal contributing to a coordinated suppression of both cytoplasmic and mitochondrial protein synthesis during transitions from active to anoxia-induced quiescent states.

Biochemical Correlates of Estivation Tolerance in the Mountainsnail Oreohelix (Pulmonata: Oreohelicidae).

Biochemical changes occurring over 7 months of estivation were studied in two species of land snail, Oreohelix strigosa (Gould) and O. subrudis (Reeve), to determine whether differential mortality during estivation is related to different energetic strategies. Laboratory-maintained snails, which were fed ad libitum prior to estivation, were compared with snails collected from the field and induced to estivate without augmenting their energy reserves. In all groups, polysaccharide was catabolized early in estivation, and protein was the primary metabolic substrate after polysaccharide reserves were depleted. Lipid was catabolized at a low rate throughout estivation. Rates of catabolism were largely statistically equivalent between species. Urea and purine bases accumulated during estivation as a result of protein catabolism, with the former being quantitatively more important. In both laboratory-maintained and field-collected snails, the rate of urea accumulation was greater in O. subrudis, resulting in higher tissue urea contents in this species at the end of the 7-month experiment. The tissue concentrations of urea at 7 months ranged from about 150 to 300 mM and were positively correlated (r = 0.99, P = 0.006) with mortality in these snails. Methylamine compounds, a class of compounds that can offset disruptive effects of elevated urea, were measured in one group of O. strigosa at 7 months of estivation and found to be low relative to urea levels. We suggest, therefore, that in the absence of elevated levels of counteracting compounds, urea may reach toxic levels and may be one factor limiting the duration of estivation that is survived by these land snails.

Acute blockage of the ubiquitin-mediated proteolytic pathway during invertebrate quiescence.

Many organisms withstand adverse environmental conditions by entering a reversible state of quiescence that may last for months or years. In this report we provide evidence that the reduction in adenylate energy status and the associated intracellular acidosis occurring during anoxia-induced quiescence combine to inhibit, directly or indirectly, the initial step in the ubiquitin-mediated proteolytic pathway in embryos of the brine shrimp Artemia franciscana. The levels of ubiquitin-conjugated proteins drop to 37% of control (aerobic) values during the first hour of anoxia and reach 7% in 24 h. ATP falls to 5% of control values under anoxia, and AMP rises reciprocally. This energy limitation is accompanied by a simultaneous depression of intracellular pH (pHi). By comparison, when embryos are subjected to artificial acidosis under aerobic conditions (pHi drops sharply, but ATP does not change for hours), ubiquitin-conjugated proteins decline to 58% after 1 h. Thus, while the proximate mechanism for the suppression of ubiquitination has not been proven, alterations in the adenylate pool and the decrease in pHi both appear to contribute to the suppression of ubiquitination. Western blot analysis indicates that the decline in ubiquitin-conjugated protein is rapidly reversed on return of embryos to control conditions. We conclude that this arrest of ubiquitination likely serves to suppress ubiquitin-mediated degradation of protein, thereby preserving macromolecular integrity and potentially explaining the remarkable extension of protein half-life observed under anoxia in these embryos.

Global arrest of translation during invertebrate quiescence.

Comparing the translational capacities of cell-free systems from aerobically developing embryos of the brine shrimp Artemia franciscana vs. quiescent embryos has revealed a global arrest of protein synthesis. Incorporation rates of [3H]leucine by lysates from 4-h anoxic embryos were 8% of those from aerobic (control) embryos, when assayed at the respective pH values measured for each treatment in vivo. Exposure of embryos to 4 h of aerobic acidosis (elevated CO2 in the presence of oxygen) suppressed protein synthesis to 3% of control values. These latter two experimental treatments promote developmental arrest of Artemia embryos and, concomitantly, cause acute declines in intracellular pH. When lysates from each treatment were assayed over a range of physiologically relevant pH values (pH 6.4-8.0), amino acid incorporation rates in lysates from quiescent embryos were consistently lower than values for the aerobic controls. Acute reversal of pH to alkaline values during the 6-min assays was not sufficient to return the incorporation rates of quiescent lysates to control values. Thus, a stable alteration in translational capacity of quiescent lysates is indicated. Addition of exogenous mRNA did not rescue the suppressed protein synthesis in quiescent lysates, which suggests that the acute blockage of amino acid incorporation is apparently not due to limitation in message. Thus, the results support a role for intracellular pH as an initial signaling event in translational control during quiescence yet, at the same time, indicate that a direct proton effect on the translational machinery is not the sole proximal agent for biosynthetic arrest in this primitive crustacean.

Yolk platelet degradation in preemergence Artemia embryos: response to protons in vivo and in vitro.

Alteration of intracellular pH (pHi) influences yolk platelet degradation during preemergence development in Artemia embryos. Cysts incubated for 10 h under conditions of aerobic development (aqueous medium equilibrated with 60% N2-40% O2, pHi greater than or equal to 7.9) exhibit a significant decrease in numbers of yolk platelets and platelet protein. In contrast, cysts incubated for 10 h under aerobic acidosis (60% CO2-40% O2, pHi = 6.8) show no significant decrease in numbers of yolk platelets or platelet protein. When subjected to alkaline conditions in vitro, yolk platelets release protein exponentially as a function of time. The process is essentially complete in 40 min. The extent of protein and lipid release from platelets increases markedly as pH of the medium is raised in increments from 6.3 to 8.0. Concomitant with these changes are reduction (50%) in platelet dry weight and reduction (21%) in platelet diameter. Transmission electron microscopy does not reveal major structural differences between isolated yolk platelets and those contained in hydrated embryos. The proton effects on platelet composition and size detected in vitro may explain in part the mechanism of platelet degradation observed during aerobic development and its suppression under conditions of acidic pHi.

Downregulation of cellular metabolism during environmental stress: mechanisms and implications.

Survival time of organisms during exposure to environmental stresses that limit energy availability is, in general, directly related to the degree of metabolic depression achieved. The energetic cost savings realized by the organism is a consequence primarily of the ability to depress ion pumping activities of cells, macromolecular synthesis, and macromolecular turnover. Evidence supporting the concept of channel arrest-the reduction in ion leakage across cell membranes during hypometabolic states-has highlighted the energetic benefits of limiting ATP turnover related to cellular ion homeostasis. Depression of protein synthesis results in substantial bioenergetic savings. However, when protein synthesis is arrested, the preservation of macromolecules becomes increasingly important as the duration of quiescence is extended because the cellular capacity for replenishing these components is reduced. It is likely that the rate of macromolecular degradation is a key feature that sets the upper time limit for survival during chronic environmental stress.

Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control.

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.