The effects of external osmotic pressure on the rates of development and emergence, respiration, and on changes in glycogen, glycerol, and trehalose concentrations have been studied in cysts of Artemia salina.
The only measured effect of external osmotic pressures less than 30 atm. on emergence and development is to determine the time required for the embryo to emerge from the cyst. Above this value the onset, rate, and final percent of emergence decrease. No emergence occurs at osmotic pressures greater than about 65 atm.
The oxygen consumption decreases with increased osmotic pressure, and is negligible at about 65 atm.
Several lines of evidence show that trehalose is the respiratory substrate, that most of the trehalose present in the dormant embryo is converted to glycogen and glycerol during development, and that the direction and extent of these conversions are controlled by the external osmotic pressure.
Glycerol appears to be present in at least two distinct locations in the cyst: within the embryo, and between the embryo and shell. Glycerol in the latter location is released into the medium at the time of emergence; the embryonic glycerol is rapidly metabolized after emergence.
The physiological significance of glycerol and trehalose in the emergence process is discussed.
An interesting relationship exists between the presence of free glycerol in high concentration and alterations in metabolic rate associated with the onset, duration and termination of dormancy in certain insects. This was first observed by Chino (1957, 1958) in diapausing eggs of the silkworm Bombyx mori, and by Wyatt & Kalf (1958) in diapausing pupae of the giant silkworm Hyalophora cecropia. Since then, considerable attention has been given to analysing the role of glycerol accumulation with respect to diapause (Wyatt & Meyer, 1959; Gilbert & Schneiderman, 1961; Wilhelm, Schneiderman & Daniel, 1961; Wyatt, 1961, 1962; Harvey, 1962; Chino, 1963), and to cold-hardness (Salt, 1958, 1959, 1961; Dubach, Pratt, Smith & Stewart, 1959) in insects.
Glycerol has also been found in high concentration in dormant cysts of the brine shrimp Artemia salina (Clegg, 1962), but its physiological significance is not known. Since these cysts consist of an embryo covered by a tough shell (cf. Dutrieu, 1960; Nakanishi, Iwasaki, Okigaki & Kato, 1962) the developing embryo must have some mechanism for rupturing this shell when it emerges from the cyst. It seemed possible that glycerol might be involved in shell-rupture because of its presence in high concentration, its relatively low molecular weight, and its well-known hygroscopic properties (Miner & Dalton, 1953). To test this possibility, a study was made of the relationship between external osmotic pressure, the emergence process, and carbohydrate metabolism in developing cysts of Artemia salina. Some of the results of this study will be presented here.
MATERIALS AND METHODS
Desiccated Artemia cysts were collected in 1962 by the Brine Shrimp Sales Co., Inc., Hayward, California. The cysts were washed (Clegg, 1962), sterilized with merthiolate (Provasoli & Sharaishi, 1959), rinsed with sterile distilled water, air-dried, and stored over CaCl2 in a desiccator. All of the cysts used in this study were prepared at the same time. Adults were cultured by the method of Bowen (1962) with 1 g. of liver extract (Difco) per litre of culture medium.
Procedure A (emergence studies)
A known number of cysts (15-25) was incubated with 0·3 ml. of the appropriate solution contained in each of the nine rings of a ‘Perma-Slide’ (Fisher Scientific Co.). Each slide was mounted on glass rods in a Petri dish containing 20 ml. of medium; the lid was placed in position and sealed with a wide rubber band. Incubations were carried out at 30° C. and the number of emerged embryos was determined at various times. The criterion of emergence is essentially the same as that of Jennings & Whitaker (1941): that time at which the shell splits and the embryo protrudes from the shell.
Procedure B (respiration studies)
Either 10 or 30 mg. of cysts, depending on the duration of the experiments, were placed in the main compartment of each of several Warburg flasks containing 0·2 ml. of 20 % KOH in the centre well. At zero time, 1·8 ml. of the incubation medium was added and the flasks were attached to the manometers. After 8 min. the flasks were placed in the water bath and were equilibrated for 7 min. The first reading was taken 5 min. later, that is, after the cysts had been in the medium for a total of 20 min. The respiratory quotient was determined by the ‘indirect-method’ of Warburg (Umbreit, Burris & Stauffer, 1957). All incubation media contained 1000 units of penicillin and 100 μ g. of streptomycin per ml. of medium. For chemical analysis of the medium and cysts the flask contents were filtered at the end of the incubation period using acid-washed filter paper. The cysts were rinsed quickly with 10 ml. of distilled water and transferred to glass homogenizers (Ten Broeck) for analysis. No shreds of filter-paper, which would produce faulty glycogen measurements, were transferred with the cysts.
Procedure C (chemical studies)
Between 40 and 45 mg. of cysts were incubated with 5· 0 ml. of sterile incubation medium containing antibiotics in Stender dishes of 25 ml. volume. The lids were sealed with stopcock grease after temperature equilibration at 30° C. At the end of the incubation period the cysts were collected and rinsed with distilled water before analysis. The incubation medium was collected from the first filtration and stored at − 20° C. prior to analysis, which was always carried out within 48 hr.
The cysts were fractionated for carbohydrates as described previously (Clegg, 1962) with the following changes : glycerol and trehalose were isolated from the 85 % ethanol-soluble fraction by paper chromatography and elution (Evans & Dethier, 1957; Clegg & Evans, 1961) prior to quantification by colorimetric methods for glycerol (Burton, 1957) and for trehalose (Dimler, Schaefer, Wise & Rist 1952). A polysaccharide, referred to as glycogen (cf. Clegg, 1962) was extracted from the ethanol-insoluble fraction with 30% potassium hydroxide at 100° C. for 15 min., and precipitated by adding 1·5 vol. of 95 % ethanol (Hudson, 1958). After placing at 4° C. overnight, this mixture was centrifuged and the soluble fraction discarded. The precipitate was washed twice with 95 % ethanol, dissolved in water, and then centrifuged briefly to remove flakes of suspended chitin. Aliquots of this supernatant were analysed by the anthrone method (Dimler et al. 1952) for glycogen. Lactic acid was determined by the method of Barker & Summerson (1941) on the ethanol-soluble fraction after removing the ethanol under reduced pressure.
The incubation medium was initially analysed for carbohydrates qualitatively by paper chromatography (Evans & Dethier, 1957; Clegg & Evans, 1961) after desalting by the pyridine method of Malpress & Morrison (1949). Subsequently, the amount of glycerol in the medium was measured directly by the method of Burton (1957); the high concentrations of NaCl and the antibiotics present in the medium did not interfere with this reaction.
Osmotic pressure values
Solutions of glucose, sucrose, and mannitol, of known osmotic pressures, were prepared from data given by Garner (1928) and were sterilized by autoclaving, or by passage through millipore filters. The osmotic pressures of NaCl and KC1 solutions were estimated using the formula π = mγ RT, where π is the osmotic pressure in atmospheres; m, the molality (which has been multiplied by 2 to obtain the effective concentration of ions); γ, the molal activity coefficient; R, the gas constant (0· 0821 l.atm mole−1 degree−1); T, the absolute temperature. Values for y were obtained from Robinson & Stokes (1959).
RESULTS AND DISCUSSION
Effects of NaCl concentration on development and emergence
Initial studies were carried out with solutions of NaCl in order to standardize the incubation procedure. Several groups of cysts were incubated as described in procedure A with different concentrations of NaCl, and the percentage emergence was determined with time (Fig. 1). Each point represents the average of at least 600 cysts. Molal (m) concentrations were used since they can be directly converted to osmotic pressure. These results showed that after emergence began, both the rate and the final percentage of emergence (about 82 %) were essentially the same at concentrations of NaCl up to 0· 75 m. Thus a major effect of NaCl concentration was to determine the period between immersion of the cysts in the medium and the onset of emergence. This interval will be referred to as the ‘lag period’. In 1· 0m-NaCl the onset, rate, and final percentage of emergence decreased. No emergence was observed in 2· 0m-NaCl, even after 72 hr. of incubation.
The cysts that did not yield nauplii (about 18%) in solutions of NaCl less than 1·0 m were removed after 72 hr. of incubation. About one-third of these were empty shells, but the remainder were found to contain embryos when the shell was opened with forceps. No oxygen was consumed by these cysts after 3 hr. of incubation, as measured by the Warburg technique. Furthermore, these cysts contained about the same levels of certain metabolites as did the cysts prior to immersion (cf. Table 1) in spite of having undergone 72 hr. of incubation. It appeared, therefore, that this part of the population represented embryos that were not metabolizing to any appreciable extent and, as a result, probably not undergoing development.
Since the solubility of oxygen decreases with increasing salt concentration, these results could be due to a decrease in the oxygen tension of the environment. However, no significant changes were observed when cysts were incubated in NaCl solutions that were saturated with oxygen and the incubation vessels flushed every 2 hr. with 100% oxygen. In addition, most of these cysts floated on the surface film, and were directly exposed to the gas phase. In view of these results it seemed unlikely that diffusion of oxygen through the medium could be a limiting factor.
Effects of external osmotic pressure on development and emergence
To determine whether these results were due to chemical effects of NaCl or to increases in osmotic pressure, a series ‘ of studies like those described was carried out using solutions of KC1, mannitol, sucrose, and glucose, the osmotic pressures of which had been measured or calculated. For each solute concentration a family of percentage emergence curves was obtained, very similar to those shown in Fig. 1 That is, below osmotic pressures of 30 atmospheres (1· 00m-NaCl ≃33 atm.) the final percentage emergence was always maximum (between 80 and 85 %) and the rate of emergence was the same after emergence began, regardless of the type of solute used. The only measured effect of increases in osmotic pressure up to 30 atmospheres was to lengthen the lag period. A relative measure of the lag period was now obtained from each of these curves by interpolating the time required for 50% of the viable embryos to emerge from the cyst (Jennings & Whitaker, 1941). This period of time, referred to as t50%E, was found to increase with osmotic pressure from about 8 to 30 atmospheres (Fig. 2). In view of the diverse nature of the solutes used it was felt unlikely that other parameters such as molecular size, charge, surface tension or viscosity affected emergence under these conditions. Rather, the increase observed in the lag period at the higher osmotic pressures was apparently due to a decrease in the effective concentration of water in the environment.
Related experiments were carried out by Jennings & Whitaker (1941) who found that the t50%E did not vary much over a certain range of sea-water concentrations, but it is difficult to compare their results with those given here because of greatly different experimental design.
Metabolic aspects of the lag period
(a) Oxygen consumption
The role of osmotic pressure on the duration of the lag period was examined further through a study of the oxygen consumption. Results obtained with solutions of NaCl using procedure B are shown in Fig. 3. The cysts began to consume oxygen about 30 min. after the solution was pipetted into each Warburg flask containing 30 mg. of cysts. Furthermore, the cysts consumed less oxygen as the salt concentration was increased. If lowered oxygen consumption of this population of cysts was due to a decrease in the number of cysts respiring, then a decrease in emergence rate and final percent emergence would also be expected since oxygen is an absolute requirement for emergence (cf. Dutrieu, 1960). Since emergence rate and final percentage emergence were not decreased in solutions less than 1 ·0m-NaCl (Fig. 1) this possibility does not seem very likely. It appeared, therefore, that increasing the salt concentration caused a decrease in the oxygen consumption of all the viable cysts in the population, at least in NaCl solutions less than 1 · 0 m. These, and subsequent values for oxygen consumption, have been corrected for the ‘Bunsen coefficient’ term in the flask constants (Umbreit et al. 1957) so the decrease in oxygen uptake atthe higher salt concentrations presumably does not reflect a decrease in oxygen solubility. In this connexion it was also found that the rate of oxygen consumption was not altered when 100 % oxygen was used as the gas phase.
A series of experiments was now carried out to determine the effects of solute composition and concentration on the oxygen consumption (Fig. 4). Here, the oxygen consumed by 30 mg. of cysts in each solution is expressed as a percent of that consumed by cysts in distilled water (control) during the first 4 hr. of incubation. It can be seen that the oxygen consumption decreased as the osmotic pressure increased. As observed in the emergence studies (Fig. 2) the effects of osmotic pressure did not become appreciable until pressures of about 8 atm. were reached.
Two respiratory quotient (R.Q.) measurements were made with each of the solutions used in the respiration studies (Figs. 3, 4). The R.Q.’s ranged from 0 · 94 to 1 · 02, indicating that carbohydrate was the major respiratory substrate. These results are in good agreement with the measurements of Dutrieu (1960) and Muramatsu (1960). Of more interest was the constancy of the R.Q. over a wide range of osmotic pressure indicating that there was no switch to lipid or protein oxidation.
(b) Chemical changes
These results suggested an important role for carbohydrate metabolism in the emergence process. About 98 % of the total carbohydrate of these cysts, excluding chitin, consists of the disaccharide trehalose, glycerol, and a polysaccharide similar to glycogen (Dutrieu, 1960; Clegg, 1962). While trehalose constitutes up to 17% of the cyst weight, little if any is present in the newly emerged nauplius, suggesting that this sugar might be a respiratory substrate. Sussman (1961) has shown that trehalose serves such a function during the germination of Neurospora ascospores and it is likely that this is a general metabolic feature of several other dormant organisms during the transition from dormant to active state (Clegg & Filosa, 1961; and also unpublished observations). Accordingly, a study of trehalose and other carbohydrates was now made in order to obtain more information on their respective roles during the lag period and how these are affected by osmotic pressure.
Cysts were incubated by procedure B. After periods of 4, 8 and 14 hr. they were removed and the carbohydrates analysed. The results of experiments using 0 · 25m-NaCl are shown in Table 1. Zero-time values represent the levels present in the desiccated cysts and any changes occurring during the incubation period are referred to this base-line. The oxygen consumption was also measured and has been converted to glucose equivalents (Umbreit et al. 1957) to enable stoichiometric comparison. It can be seen that glycerol and glycogen increased with incubation time, while trehalose decreased markedly. The column at the right attempts to account for the decrease in trehalose by increases in glycogen, glycerol, and glucose equivalents of the amount of oxygen consumed. A value of one in this column would suggest that most of the trehalose was converted to glycerol and glycogen, while the remainder was oxidized. The results are in good agreement with this suggestion. They also show that glycogen cannot be the respiratory substrate as suggested by Muramatsu (1960, 1961) who failed to detect trehalose in extracts of Artemia cysts. Not given in this table are the insignificant amounts of lactic acid measured in the dry cysts ( < 0·3 μg./mg. dry weight). This low level did not change with time or osmotic pressure.
To determine the effects of osmotic pressure on the levels of these metabolites, several groups of cysts were incubated for 4 hr. in 0·25, 0·50, 0·75 and 1·00m-NaCl according to procedure B, and then analysed as described. In Fig. 5 the changes in the concentrations of these substances and in the amount of oxygen consumed are expressed as a percentage of the change occurring in cysts incubated with 0·25 m-NaCl. In order to correlate these changes with development, I included in Fig. 5 the reciprocal of t50%E which is a measure of the rate of development based on the time required for emergence. On this basis the rate of development decreased uniformly as the osmotic pressure increased. Very closely correlated with this decreasing developmental rate were decreases in the rate of trehalose utilization and oxygen consumption, indicating that trehalose oxidation was directly connected with development, probably as an energy source. Compared to these the synthesis of glycogen decreased more markedly with increased osmotic pressure. In contrast, the net synthesis of glycerol actually increased, and the extent of this increase was related to the decrease in glycogen over the entire range of osmotic pressure studied. This further supports the suggested partitioning of trehalose between the synthesis of glycogen and glycerol and, in addition, shows that the direction of synthesis depends on the magnitude of osmotic pressure.
Relationship between cyst glycerol, medium glycerol, emergence and external osmotic pressure
The stimulation of net glycerol synthesis by increased osmotic pressure indicated that free glycerol might play a role in overcoming the osmotic pressure difference between the interior of the cyst and its environment. Additional support for this view was the finding that free glycerol appeared in the medium only after emergence had occurred. This qualitative observation was examined further by incubating the cysts under sterile conditions as described in procedure C, and then analysing the medium and the cysts for glycerol content after appropriate incubation periods.
The results, given in Fig. 6, include percentage emergence data which have been taken directly from Fig. 1. The level of free glycerol in the desiccated cysts (33·8 μ g./mg. dry weight) was designated as zero at the beginning of each experiment to aid in interpreting the data.
Maximum cyst glycerol was always reached at about the time that emergence began, and the extent of this maximum increased with osmotic pressure (Fig. 6). Therefore, the net synthesis of glycerol occurred only during the lag period when the embryo was completely enclosed by the shell. Following this maximum, cyst glycerol decreased rapidly. Although some of this decrease is due to the release of glycerol into the medium (Fig. 6), most of the glycerol is retained in the emerging nauplius where it then rapidly disappears (Clegg, 1962). The net effect of these two processes results in the essentially complete loss of free glycerol after emergence.
It can also be seen in Fig. 6 that glycerol did not appear in the medium until emergence began and that its increase was very closely correlated with the time course of the percentage emergence. These results indicated that glycerol was released into the medium only when each embryo emerged from its shell. The only apparent alternative would be the gradual release or leaching of glycerol into the medium from the emerged nauplius, and this has been ruled out by the following experiment.
Newly emerged nauplii were washed and incubated in fresh sterile solutions of NaCl. Glycerol concentrations in the medium and nauplii were measured at zero time and after 6 hr. of incubation at 30 ° C. During this period the nauplius glycerol concentration decreased by about 30 μ g./mg. dry weight, while no glycerol appeared in the medium (limit of detection = 0 · 5 μ g./mg. dry weight). This rapid metabolism of glycerol after emergence is especially interesting because it suggests that the mechanisms leading to net glycerol synthesis are operative only when the embryo is enclosed by the shell.
Location and significance of glycerol in the cyst
Each cyst is composed of an embryo surrounded by two membranes and a shell. At emergence the outer membrane and shell break and the embryo, still covered by the inner membrane, protrudes from the cyst (Lochhead, 1941; Tha Myint, 1956; Dutrieu, 1960; Nakanishi et al. 1962). Accordingly, any fluid contained in the extra-embryonic spaces will be released at this time. Since glycerol suddenly appears in the medium at emergence (Fig. 6) a reasonable interpretation would be that it originates in the contents of these extra-embryonic spaces.
If free glycerol in the cyst is functioning osmotically during the lag period, and thereby enabling the embryo to emerge, then the osmotic pressure exerted by this glycerol can be expected to bear some relation to the external osmotic pressure. In order to evaluate this suggestion, it was necessary to estimate the water content of the hydrated cysts. Dry cysts were incubated in distilled water for 8 hr., filtered, weighed, and then dried to constant weight (Clegg, 1962). The average value obtained for over 800 cysts was approximately 0 · 5 μ l. of water/mg. of cysts, or about 50% of the total hydrated cyst weight. Assuming this water to be the effective solvent for all of the cyst glycerol, then the internal osmotic pressure due to glycerol can be estimated and compared with the known osmotic pressure of the environment in which the cysts were incubated (Table 2). When the cyst glycerol reached its maximum concentration, it exerted an internal osmotic pressure nearly twice that of the environment. Admittedly, some of the assumptions used to obtain the values for internal osmotic pressure due to glycerol are subject to question. However, even if the real values are actually 50 % lower than those given (which would mean that the water content would equal the entire cyst weight) it is still possible at least to equalize the internal and external osmotic pressures solely on the basis of glycerol concentration in the cysts. But the interpretation of these calculations need not depend upon absolute values for internal osmotic pressure. This is shown in the columns headed μ π in Table 2 which represent the increases in internal and external osmotic pressures as the concentration of the external medium is increased. It can be seen that increases produced in the external osmotic pressure are always less than the corresponding increases which occurred in the internal osmotic pressure on the basis of glycerol accumulation in the cyst. These calculations are highly suggestive of an osmotic role for glycerol in the rupture of the shell.
On the roles of trehalose and glycerol during development
The following discussion summarizes the sequence of events that is suggested to occur during the transition of the dormant encysted embryo to the emerged nauplius.
From about 8 to 30 atm., the rates of development and aerobic metabolism decrease as the external osmotic pressure increases (Figs. 1 − 4). It is proposed that this results initially from a deficiency of water in the cysts which becomes more severe as the osmotic pressure is increased up to about 65 atm. where no water is imbibed and no development occurs. Although direct evidence for this water deficiency is lacking, it is difficult to imagine an initial effect of increased osmotic pressure other than to cause a decrease in the availability of water or possibly oxygen, and it should be recalled that the latter possibility is not a very likely one.
One effect of this water deficiency is to cause a low rate of trehalose oxidation. This would lead to an inability to meet the energy requirements for a maximal rate of development which, in turn, could account for the observed increase in the duration of the lag period with increases in the external osmotic pressure (Fig. 1). Such an interpretation is justified for the following reasons : first, trehalose appears to be the only respiratory substrate (R.Q. studies, Table 1); secondly, the rates of development, trehalose breakdown, and oxygen consumption correlate with each other very closely over a wide range of osmotic pressure (Fig. 5); thirdly, the absence of lactic acid accumulation with incubation time and increased osmotic pressure indicates that anaerobic energy-yielding pathways leading to lactic acid formation are not operating. Since the synthesis of glycerol from trehalose probably requires energy (Wilhelm & Schneiderman, 1961), this conversion apparently does not supply the energy requirement. Therefore, unless other means are available, it would appear that the complete oxidation of trehalose is the major, and perhaps the only, source of energy for development. It can be expected that anything which sufficiently decreases the supply of energy will also slow development and, therefore, the time required for emergence to occur.
Another effect of increased external osmotic pressure is to increase the level of free glycerol in the cysts (Fig. 6). This effect is due to the observed increase in the conversion of trehalose to glycerol at the higher external osmotic pressures when trehalose oxidation is decreased (Table 1, Fig. 5, R.Q. studies). Net glycerol synthesis might occur through an enzymic reduction of dihydroxyacetone, or its phosphate ester, followed by a phosphatase. Such a system has previously been shown to be present in certain diapausing insects where free glycerol also accumulates (Chino, 1958, 1963; Wyatt & Meyer, 1959; Wyatt, 1962). There are other possibilities, such as the direct reduction of glyceraldehyde, or changes in the activity of enzymes directly involved with the metabolism of glycerol (Lin, Levin & Magasanik, 1960). Lipid hydrolysis cannot account for glycerol accumulation in Artemia cysts since no change has been observed in the ‘total lipid fraction’ during the lag period (Urbani, 1959). Moreover, the amount of glycerol to be accounted for (Fig. 6) would require an enormous amount of glyceride— greater than twice the weight of the entire cyst, depending on the assumptions used for the estimate.
It is further proposed that the gradual accumulation of free glycerol in the cyst produces a corresponding increase in the internal osmotic pressure, resulting in an increased ability of the cyst to imbibe water and continue its development. An osmotic rupturing of the shell would then occur if the extent of this increase in the internal osmotic pressure eventually exceeded that of the environment. The calculations given in Table 2 suggest that this possibility is not unlikely.
The ecological significance of this feedback-type of system is that it enables the embryo to develop and emerge successfully over a wide range of external osmotic pressures. Such an adaptation is of great advantage to an organism such as Artemia which normally lives in environments ranging from very dilute sea water to concentrated brines.
This study was supported by grant GM 10673 from the U.S.P.H.S. I express my thanks to Dr Howard Lenhoff for a critical reading of the manuscript and many helpful discussions.