The Role of Glycerol and Inorganic Ions in Osmoregulatory Responses of the Euryhaline Flagellate Chlamydomonas pulsatilla Wollenweber

The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [¹⁴C] mannitol and ³H₂O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [¹⁴C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (π_int) matched closely the external osmolality (π_ext) over the entire salinity range except at 10% ASW where π_int exceeded π_ext by 120 to 270 milliosmoles per kilogram H₂O.     

Structural Confirmation of a Unique Carotenoid Lactoside,P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3′S,5′R,6′S)‐13′‐cis‐5,6‐epoxy‐3′,5′‐dihydroxy‐3‐(β‐d ‐galactosyl‐(1→4)‐β‐d ‐glucosyl)oxy‐6′,7′‐didehydro‐5,6,7,8,5′,6′‐hexahydro‐β,β‐caroten‐20‐al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free‐living peridinin‐containing dinoflagellates and marine invertebrates that harbor peridinin‐containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin‐containing dinoflagellates. Fucoxanthin‐containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin‐containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.     

TRANSFER OF PRODUCTS BETWEEN EPIPHYTIC MARINE ALGAE AND HOST PLANTS1,2

The red alga Smithora naiadum is normally found only as an epiphyle on the sea grasses Phyllospadix scouleri and Zostera marina. I used ³²P and ¹⁴CO₂ to examine the chemical communication between host and alga. Both ³²P and the product of ¹⁴CO₂ light fixation moved from the host to the alga. Reverse movement between host and epiphyte was also demonstrated. Part of this transfer occurred through the plant and part occurred by leakage from the host into the medium and subsequent uptake by the alga. Although plants were initially labeled in the light, transfer of ¹⁴C was light independent. Transfer of ¹⁴C-labeled products between host and epiphyte was also shown for Punctaria orbiculata and Z. marina; for Microdadia coulteri on Grateloupia doryphora, and between Gonimophyllum skottsbergii and Botryoglossum ruprechtiana. Epiphyte-host associations do not require a penetrating rhizoid for an exchange of the isotopes tested. By their proximity alone, epiphytic flora are apparently capable l exchanging products before these are diluted by the sea.     

Do the Anemonefish Amphiprion ocellaris (Pisces: Pomacentridae) Imprint Themselves to Their Host Sea Anemone Heteractis magnifica (Anthozoa: Actinidae)?

Juvenile anemonefishes detect their host sea anemone by olfactory stimuli; in order to investigate whether this behaviour is innate or acquired, the anemonefish species Amphiprion ocellaris was bred in two different ways: 1. With no host sea anemone present at all (–A); and 2. With the specific host sea anemone Heteractis magnifica present in the hatching aquarium, so that these eggs were laid and hatched close to the sea anemone, as in nature (+A). The two different types of juvenile A. ocellaris were presented to the odours of the host sea anemone H. magnifica in two sets of short-term experiments with the host (a) visually hidden in a net cage, and (b) visible but physically separated from the anemonefishes. In both cases, a water flow was established between fishes and host. The +A-fishes found their host by olfactory and not by visual stimuli. In both series, the –A-fishes showed a significantly lower affinity behaviour towards the odour compounds from the host sea anemone than the +A-fishes did. A third type of experiment was a direct confrontation between fishes and host; here, the –A-fishes were indifferent towards the host sea anemone for almost 48 h, while the +A-fishes acclimated to the host sea anemone within the first 5 min of the direct confrontation. The results of this study suggest that Amphiprion ocellaris imprints itself olfactorily to its species-specific host sea anemone Heteractis magnifica, and, furthermore, may be genetically disposed towards olfactory recognition of the host sea anemone.     

The influence of "host release factor" on carbon release by zooxanthellae isolated from fed and starved Aiptasia pallida (Verrill).

Symbiotic dinoflagellates (zooxanthellae) typically respond to extracts of host tissue with enhanced release of short-term photosynthetic products. We examined this "host release factor" (HRF) response using freshly isolated zooxanthellae of differing nutritional status. The nutritional status was manipulated by either feeding or starving the sea anemone Aiptasia pallida (Verrill). The release of fixed carbon from isolated zooxanthellae was measured using 14C in 30 min experiments. Zooxanthellae in filtered seawater alone released approximately 5% of photosynthate irrespective of host feeding history. When we used a 10-kDa ultrafiltrate of A. pallida host tissue as a source of HRF, approximately 14% of photosynthate was released to the medium. This increased to over 25% for zooxanthellae from anemones starved for 29 days or more. The cell-specific photosynthetic rate declined with starvation in these filtrate experiments, but the decline was offset by the increased percentage release. Indeed, the total amount of released photosynthate remained unchanged, or even increased, as zooxanthellae became more nutrient deficient. Similar trends were also observed when zooxanthellae from A. pallida were incubated in a 3-kDa ultrafiltrate of the coral Montastraea annularis, suggesting that HRF in the different filtrates operated in a similar manner. Our results support the suggestion that HRF diverts surplus carbon away from storage compounds to translocated compounds such as glycerol.     

Characterization of cell ion exchange in the sea anemoneCondylactis gigantea

Summary Maintenance of intracellular ion contents and their relations to transmembrane potential were studied in tentacles ofCondylactis gigantea. Tentacles leached at 2°C in 10 mMK⁺ and 2 mM K⁺ artificial seawaters (10K ASW and 2K ASW) with and without 2 MM ouabain, and in 0K ASW, lost cell K⁺ and gained Na⁺. Rewarming to 25°C in 10K ASW resulted in a marked accumulation of K⁺ and extrusion of Na⁺ in tentacles leached in 10K ASW and in 0K ASW. Initial rate of Na⁺ extrusion was twice the initial rate of K⁺ accumulation, suggesting a pump coupling ratio of 2. In tentacles leached and rewarmed in 2K ASW, no net reaccumulation of K⁺ and little net extrusion of Na⁺ was observed; i.e., the pump just kept pace with the leaks. Ouabain inhibited K⁺ reaccumulation and Na⁺ extrusion. This effect was less marked in 10K ASW than in 2K ASW confirming, in anemone tentacles, the well documented ouabain-K⁺ antagonism observed in other systems. In no case did K _i ⁺ /K ₀ ⁺ equal Cl ₀ ^– /Cl _i ^– ; therefore, the distribution of these ions did not fit a Donnan distribution. Transmembrane potential difference was –22±3 mV in 10K ASW at 25°C. It fitted a modified Nernst equation which includes the pump coupling ratio and a Na⁺ to K⁺ permeability ratio of 0.31.A moderately high permeability of the cell membrane to Na⁺, and a ouabain and K⁺ sensitive ion pump, exchanging 2 Na⁺ for 1 K⁺, appear to be responsible for the observed ionic distribution and transmembrane potential in anemone cells.     

Cloning and Characterization of ApRab4, a Recycling Rab Protein of Aiptasia pulchella, and Its Implication in the Symbiosome Biogenesis

The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin–avidin detection system, we found evidence for symbiosome–endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome.     

Nutrient cycling in early coral life stages: Pocillopora damicornis larvae provide their algal symbiont (Symbiodinium) with nitrogen acquired from bacterial associates

The waters surrounding coral reef ecosystems are generally poor in nutrients, yet their levels of primary production are comparable with those reported from tropical rain forests. One explanation of this paradox is the efficient cycling of nutrients between the coral host, its endosymbiotic alga Symbiodinium and a wide array of microorganisms. Despite their importance for the animals' fitness, the cycling of nutrients in early coral life stages and the initial establishment of partnerships with the microbes involved in these processes has received little scrutiny to date. Nitrogen is an essential but limited nutrient in coral reef ecosystems. In order to assess the early nutrient exchange between bacteria and corals, coral larvae of the species Pocillopora damicornis were incubated with two coral‐associated bacteria (Alteromonas sp., or Vibrio alginolyticus), prelabeled with the stable nitrogen isotope ¹⁵N. The incorporation and translocation of nitrogen from Vibrio‐ and Alteromonas bacteria into P. damicornis coral larvae and specifically into the coral‐symbiotic Symbiodinium were detected by nanoscale secondary ion mass spectrometry (NanoSIMS). A significant increase in the amount of enriched ¹⁵N (two to threefold compared to natural abundance) was observed in P. damicornis larvae within 8 h of incubation for both bacterial treatments (one‐way ANOVA, F_5,53 = 18.03, P = 0.004 for Alteromonas sp. and F_5,53 = 18.03, P = 0.0001 for V. alginolyticus). These findings reveal that coral larvae acquire nutrients previously taken up from the environment by bacteria. The additional nitrogen may increase the survival rate and fitness of the developing coral and therefore contribute to the successful maintenance of coral reefs.     

Cover

Loss (bleaching) of symbiotic dinoflagellates (genus Symbiodinium) from the sea anemone Aiptasia pallida caused by elevated temperatures or disruption of symbiont photosynthesis can be restored through exposure to axenic Symbiodinium cultures (top part of figure; Symbiodinium appear red due to chlorophyll autofluorescence under blue light). [Vol. 49, No. 3, pp.447–458]     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

The Relationship between Inorganic Nitrogen Metabolism and Proline Accumulation in Osmoregulatory Responses of Two Euryhaline Microalgae

Chlorella autotrophica, a euryhaline marine alga, and Stichococcus bacillaris, a salt-tolerant soil alga, grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining high levels of NADPH-glutamate dehydrogenase. Nitrate reductase showed no change in MSX-adapted cells. For both species, MSX-adapted cells retained their capacity to accumulate proline in response to salinity, and in S. bacillaris no major shift was observed in the presence of MSX toward the accumulation of sorbitol. Following transfer from 33 to 150% artificial seawater (ASW), both algae exhibited increases in organic solute levels without a lag. Within 6 h of this sudden increase in salinity, the levels of proline in C. autotrophica and of proline and sorbitol in S. bacillaris were similar to those found in steady state 150% ASW cultures. Following transfer from 33 to 150% ASW, S. bacillaris continued [¹⁴C] bicarbonate photoassimilation at a normal rate and maintained active enzymes of nitrogen assimilation. The incorporation of [¹⁴C]phenylalanine into proteins was inhibited for about 30 minutes in MSX-free cells and 90 minutes in MSX-adapted cells following transfer from 33 to 150% ASW; the recovery after these lag periods was almost complete.     

Effects of anemonefish on giant sea anemones: expansion behavior,growth, and survival

The symbiosis between giant sea anemones and anemonefish on coral reefs is well known, but little information exists on impacts of this interaction on the sea anemone host. On a coral reef at Eilat, northern Red Sea, individuals of the sea anemone Entacmaea quadricolor that possessed endemic anemonefish Amphiprion bicinctus expanded their tentacles significantly more frequently than did those lacking anemonefish. When anemonefish were experimentally removed, sea anemone hosts contracted partially. Within 1–4 h in most cases, individuals of the butterflyfish Chaetodon fasciatus arrived and attacked the sea anemones, causing them to contract completely into reef holes. Upon the experimental return of anemonefish, the anemone hosts re-expanded. The long-term growth rate and survival of the sea anemones depended on the size and number of their anemonefish. Over several years, sea anemones possessing small or no fish exhibited negative growth (shrinkage) and eventually disappeared, while those with at least one large fish survived and grew. We conclude that host sea anemones sense the presence of symbiotic anemonefish via chemical and/or mechanical cues, and react by altering their expansion behavior. Host sea anemones that lack anemonefish large enough to defend them against predation may remain contracted in reef holes, unable to feed or expose their tentacles for photosynthesis, resulting in their shrinkage and eventual death.     

Research note: Induction of gamete discharge by hypertonic treatment in the green alga Bryopsis plumosa (Caulerpales,Chlorophyta)

Brief treatment with hypertonic solutions induced gamete discharge from gametangia in the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh. It is known that gamete discharge in this alga is triggered by a change from darkness to light. However, in this study mature gametangia, incubated for 30–120 s in artificial seawater (ASW) with an additional 0.4–0.6 M NaCl and then transferred into pure ASW, discharged gametes in darkness. The treatment did not affect the motility of the gametes. Addition of sucrose (1.0–1.2 M) to ASW also induced gamete discharge in darkness. Similar results were obtained by adding KCl (0.4 M) or mannitol (1.2 M) to ASW. Continuous incubation of gametangia in such hypertonic solutions also induced gamete discharge but led to a delay and a reduction in the rate of gamete discharge, and a loss of gamete motility. In gametangia treated with the hypertonic solutions, as well as in those exposed to light, shortening of the gametangial length was observed before gamete discharge.     

Cloning and Characterization of the First Cnidarian ADP-Ribosylation Factor,and Its Involvement in the <Emphasis Type="Italic">Aiptasia-Symbiodinum</Emphasis> Endosymbiosis

Marine cnidarian-microalgal endosymbiosis is a form of intracellular association that contributes greatly to the high primary productivity of reefs; however, little is known about its molecular mechanisms. Since the ADP-ribosylation factor (ARF) family proteins are key regulators of host intracellular vesicle transport systems, which are critical to many endosymbiotic interactions, we set out to clone and characterize ARF proteins in the symbiotic sea anemone Aiptasia pulchella. Experiments indicated that at least 3 ARF protein classes (class I, class II and class III) were present and expressed as a single messenger RNA species in Aiptasia, with highest mRNA expression levels for apARF1, medium for apARF5, and lowest for apARF6. Quantitative analysis revealed a great reduction at both the RNA and the protein levels in apARF1, but not apARF5 and apARF6, in the symbiotic animals. The apARF1 protein was highly homologous in sequence to other known ARF1 proteins and displayed a Golgi-like localization pattern. Overall, our study identified apARF1 as a potential negative regulator of Aiptaisia-microalgal endosymbiosis.     

Parasitic anemone infects the invasive ctenophore <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis> in the North East Atlantic

We report of the first finding of parasitic sea anemone larvae infecting the invasive ctenophore Mnemiopsis leidyi in the North East Atlantic. Parasitic anemone larvae are common in the native habitat of Mnemiopsis, but have not previously been reported from any of the locations where Mnemiopsis has been introduced. General morphology and 18S rRNA sequences support the identification of the larvae as Edwardsiella sp. Excised anemone larvae were reared through metamorphosis and confirmed the identification of the parasite. Both Mnemiopsis and Edwardsiella larvae were monitored weekly during 2007 and 2008 and parasitic larvae were recorded from September to November both years. The highest density was observed in September 2008 when Edwardsiidae larvae reached 2.3 ind m⁻³. In 2008 total density of the parasite occasionally exceeded 40% of the host density and the potential consequences for Mnemiopsis population dynamics are discussed.     

CHANGES IN THE ULTRASTRUCTURE OF SYMBIOTIC ZOOXANTHELLAE (SYMBIODINIUM SP., DINOPHYCEAE) IN FED AND STARVED SEA ANEMONES MAINTAINED UNDER HIGH AND LOW LIGHT1

The ultrastructure of symbiotic dinoflagellates (Symbiodinium sp., zooxanthellae) in the sea anemone Aiptasia pallida Verrill was examined in well-fed or starved (up to 120 days) anemones maintained under two light levels (5 and 50 μmol · m^?2· s^?1). Cell size of zooxanthellae was not affected by feeding history; however, both light and feeding history affected the relative cell volume of chloroplasts, lipids, and vacuoles. Stereological analysis of transmission electron micrographs showed that algae in low-light starved anemones had 10 times as much lipid (17.4% of cell volume) as those in well-fed anemones under the same light conditions (1.8%). The lipid content of algae from anemones in high light increased from 15.4% in well-fed anemones to 30.1% in starved anemones. The starch content of zooxanthellae in low-light anemones was law (4.1%) and not affected by feeding history, while the starch content of zooxanthellae in high-light anemones was greater (10.7%), with some differences among groups. Algal photoacclimation to low light included an increase in chloroplast relative volume from 17% (in well-fed high-light anemones) to 33% in well-fed low-light anemones. Starvation of the host resulted in a significant decrease in chloroplast volume in zooxanthellae in anemones at both light levels. Morphometry provides quantitative confirmation of biochemical and physiological data on zooxanthellae, because the changes in zooxanthellae with starvation of the host are consistent with other indicators of nutrient limitation of zooxanthellae of A. pallida held without food for long periods of time.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

ADAPTATION OF THE UNICELLULAR ALGA DUNALIELLA PARVA TO A SALINE ENVIRONMENT1

The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴CO₂ incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴C sorbitol and ³H₂O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell-free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol. These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.