Symbiotic Dinoflagellate Algae Research Articles

A candidate transporter allowing symbiotic dinoflagellates to feed their coral hosts

The symbiotic partnership between corals and dinoflagellate algae is crucial to coral reefs. Corals provide their algal symbionts with shelter, carbon dioxide and nitrogen. In exchange, the symbiotic algae supply their animal hosts with fixed carbon in the form of glucose. But how glucose is transferred from the algal symbiont to the animal host is unknown. We reasoned that a transporter resident in the dinoflagellate cell membrane would facilitate outward transfer of glucose to the surrounding host animal tissue. We identified a candidate transporter in the cnidarian symbiont dinoflagellate Breviolum minutum that belongs to the ubiquitous family of facilitative sugar uniporters known as SWEETs (sugars will eventually be exported transporters). Previous gene expression analyses had shown that BmSWEET1 is upregulated when the algae are living symbiotically in a cnidarian host by comparison to the free-living state [1, 2]. We used immunofluorescence microscopy to localise BmSWEET1 in the dinoflagellate cell membrane. Substrate preference assays in a yeast surrogate transport system showed that BmSWEET1 transports glucose. Quantitative microscopy showed that symbiotic B. minutum cells have significantly more BmSWEET1 protein than free-living cells of the same strain, consistent with export during symbiosis but not during the free-living, planktonic phase. Thus, BmSWEET1 is in the right place, at the right time, and has the right substrate to be the transporter with which symbiotic dinoflagellate algae feed their animal hosts to power coral reefs.

Colony self-shading facilitates Symbiodiniaceae cohabitation in a South Pacific coral community

The ecological success of tropical corals is regulated by symbiotic dinoflagellate algae (Symbiodiniaceae). Corals can associate with multiple Symbiodiniaceae species simultaneously, yet the conditions that permit Symbiodiniaceae cohabitation are not understood. We examined how corals self-shade their own tissues causing within-colony light gradients that drive Symbiodiniaceae photoacclimatory processes and positional genetic disparity. Paired light ‘exposed’ and ‘shaded’ samples from 20 coral species were collected from a shallow coral reef (Rarotonga, Cook Islands). Through active chlorophyll fluorometry, rapid light curves revealed that exposed Symbiodiniaceae exhibited 50% higher values in minimum saturating irradiances and demonstrated a shift towards preferential nonphotochemical quenching [1 – Q], consistent with higher overall light exposure. High-throughput or targeted DNA sequencing of ITS2 and psbAncr markers demonstrated that corals harboured distinct and/or differentially abundant Symbiodiniaceae ITS2 sequences (typically rare in relative abundance) or multiple ITS2 intragenomic variant profiles across shaded vs exposed regions. In Hydnophora cf. microconos, within-colony symbiont genetic disparity was positively correlated with the magnitude of difference in [1 – Q] utilisation. Together, these results suggest that within-colony light gradients produce distinct optical niches that enable symbiont cohabitation via photoadaptation, a phenomenon that is expected to increase the adaptive capacity of corals under future climates.

Polystyrene nanoplastics impair the photosynthetic capacities of Symbiodiniaceae and promote coral bleaching

Reef-building corals are increasingly threatened by global and regional stresses, which affect the stability of the coral-Symbiodiniaceae association. Among them, plastic pollution has been an ongoing and growing concern. Whereas several studies have highlighted the detrimental impact of microplastics (0.1 μm–5 mm) on corals and their symbiotic dinoflagellate algae, the physiological changes induced by nanoplastic (NP, <0.1 μm) pollution are still poorly known. Long-term experiments (4 weeks) were conducted to investigate the effects of ecologically relevant NP concentrations (0 to 0.5 mg/L of 20 nm polystyrene NPs) on two Symbiodiniaceae in culture [CCMP2467 or Clade A1 and pd44b or Clade F1]. The effects of 0.5 mg/L NPs were also evaluated on Clade A1 living in symbiosis with the coral Stylophora pistillata, to assess the in hospite effects of NPs on coral symbionts. The photosynthetic efficiency of photosystem II, the oxidative status of the Symbiodiniaceae and the coral host, as well as the host-symbiont stability were evaluated at the end of the experiment. Symbiodiniaceae in culture exhibited a significant decrease in the maximal electron transport rate (ETRmax) at NP concentrations as low as 0.005 mg/L, highlighting an impairment of the photosynthetic capacities of the dinoflagellates in presence of nanoplastics. Also, Clade A1 exhibited a significant decrease in its Total Antioxidant Capacity (TAC) and an increase in Lipid Peroxidation (LPO), which evidence oxidative stress and cellular damage. Interestingly, Clade A1 in hospite did not show any signs of oxidative stress, however, the coral host exhibited increased TAC and LPO. Additionally, exposure of S. pistillata to 0.5 mg/L NPs induced significant bleaching (loss of symbionts and photosynthetic pigments). Overall, NPs were detrimental for both the Symbiodiniaceae in culture and the host-symbiont association. In the future, the persistence of reef corals may be severely impacted by the cumulative effects of nanoplastic pollution along with global warming.

Lineage dynamics of the endosymbiotic cell type in the soft coral Xenia

Many corals harbour symbiotic dinoflagellate algae. The algae live inside coral cells in a specialized membrane compartment known as the symbiosome, which shares the photosynthetically fixed carbon with coral host cells while host cells provide inorganic carbon to the algae for photosynthesis1. This endosymbiosis—which is critical for the maintenance of coral reef ecosystems—is increasingly threatened by environmental stressors that lead to coral bleaching (that is, the disruption of endosymbiosis), which in turn leads to coral death and the degradation of marine ecosystems2. The molecular pathways that orchestrate the recognition, uptake and maintenance of algae in coral cells remain poorly understood. Here we report the chromosome-level genome assembly of a Xenia species of fast-growing soft coral3, and use this species as a model to investigate coral–alga endosymbiosis. Single-cell RNA sequencing identified 16 cell clusters, including gastrodermal cells and cnidocytes, in Xenia sp. We identified the endosymbiotic cell type, which expresses a distinct set of genes that are implicated in the recognition, phagocytosis and/or endocytosis, and maintenance of algae, as well as in the immune modulation of host coral cells. By coupling Xenia sp. regeneration and single-cell RNA sequencing, we observed a dynamic lineage progression of the endosymbiotic cells. The conserved genes associated with endosymbiosis that are reported here may help to reveal common principles by which different corals take up or lose their endosymbionts.

Symbiont photosynthesis in giant clams is promoted by V-type H+-ATPase from host cells.

Giant clams (genus Tridacna) are the largest living bivalves and, like reef-building corals, host symbiotic dinoflagellate algae (Symbiodinium) that significantly contribute to their energy budget. In turn, Symbiodinium rely on the host to supply inorganic carbon (Ci) for photosynthesis. In corals, host 'proton pump' vacuolar-type H+-ATPase (VHA) is part of a carbon-concentrating mechanism (CCM) that promotes Symbiodinium photosynthesis. Here, we report that VHA in the small giant clam (Tridacna maxima) similarly promotes Symbiodinium photosynthesis. VHA was abundantly expressed in the apical membrane of epithelial cells of T. maxima's siphonal mantle tubule system, which harbors Symbiodinium Furthermore, application of the highly specific pharmacological VHA inhibitors bafilomycin A1 and concanamycin A significantly reduced photosynthetic O2 production by ∼40%. Together with our observation that exposure to light increased holobiont aerobic metabolism ∼5-fold, and earlier estimates that translocated fixed carbon exceeds metabolic demand, we conclude that VHA activity in the siphonal mantle confers strong energetic benefits to the host clam through increased supply of Ci to algal symbionts and subsequent photosynthetic activity. The convergent role of VHA in promoting Symbiodinium photosynthesis in the giant clam siphonal mantle tubule system and coral symbiosome suggests that VHA-driven CCM is a common exaptation in marine photosymbioses that deserves further investigation in other taxa.

Sensitivity to oxidative stress is not a definite predictor of thermal sensitivity in symbiotic dinoflagellates

Coral bleaching, the loss of symbiotic dinoflagellate algae (genus Symbiodinium) and/or photosynthetic algal pigments from corals, is thought to be primarily triggered by the thermal dysfunction of photosynthesis and a consequent build-up of reactive oxygen species. However, different corals exhibit differential bleaching susceptibilities, perhaps resulting from dissimilar abilities to deal with oxidative stress. We therefore tested whether thermal sensitivity in Symbiodinium is correlated with the capacity to deal with oxidative stress, by comparing the effects of increased temperature and hydrogen peroxide (H2O2) concentration on the photosynthetic performance of four cultured Symbiodinium ITS2 types (A1, B2, E, and F1) and two freshly isolated Symbiodinium ITS2 types (a temperate A-type and B1). Generally, the maximum quantum yield of photosystem II (Fv/Fm) declined in Symbiodinium with both increasing thermal and oxidative stress. However, not all types followed this pattern. Cultured Symbiodinium A1 showed a strong negative response to elevated temperature but little response to the addition of H2O2, while cultured F1 showed an increase in Fv/Fm at elevated temperature but a decline in this parameter to almost zero at high H2O2 concentrations. Furthermore, both freshly isolated Symbiodinium types appeared to be relatively stress tolerant, with the temperate A-type showing an especially high resistance to both stressors. In conclusion, a range of Symbiodinium types were shown to differ in their susceptibilities to both thermal and oxidative stress, though in contrast to our original hypothesis, sensitivity to oxidative stress did not necessarily predict thermal sensitivity (or vice versa).

Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis

Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

The influence of salinity and copper exposure on copper accumulation and physiological impairment in the sea anemone, Exaiptasia pallida

Copper is a common pollutant in many aquatic environments, particularly those surrounding densely populated areas with substantial anthropogenic inputs. These same areas may also experience changes in salinity due to freshwater discharge and tidal influence. Biota that inhabit near-shore coastal environments may be susceptible to both stressors. Although copper is a noted concern in marine environments, effects of copper and varying salinity on symbiotic cnidarians are only scarcely studied. The sea anemone, Exaiptasia pallida, was used to investigate effects of copper on physiological impairment (i.e. activities of anti-oxidant enzymes) at two different salinities (20 and 25ppt). E. pallida was exposed to a control and three elevated copper concentrations for up to 21d, and copper accumulation and activity of the enzymes: catalase, glutathione reductase, glutathione peroxidase, and carbonic anhydrase were measured in the anemones. Photosynthetic parameters in E. pallida's symbiotic dinoflagellate algae were also quantified. Over the course of the exposure, E. pallida accumulated copper in a concentration-dependent manner. Higher tissue copper concentrations were observed in anemones exposed to the lower salinity water (20ppt), and physiological impairment was observed as a consequence of both increased copper exposure and decreased salinity; however, changes in salinity caused a greater response than copper exposure, at the levels tested. In general, antioxidant enzyme activity increased as a consequence of decreased salinity and/or increased copper exposure. These results clearly demonstrated the influence of two local stressors, at environmentally realistic concentrations, on a sensitive cnidarian, and highlight the importance of characterizing combined exposure scenarios.

A nanoscale secondary ion mass spectrometry study of dinoflagellate functional diversity in reef-building corals.

Nutritional interactions between corals and symbiotic dinoflagellate algae lie at the heart of the structural foundation of coral reefs. Whilst the genetic diversity of Symbiodinium has attracted particular interest because of its contribution to the sensitivity of corals to environmental changes and bleaching (i.e. disruption of coral-dinoflagellate symbiosis), very little is known about the in hospite metabolic capabilities of different Symbiodinium types. Using a combination of stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), we investigated the ability of the intact symbiosis between the reef-building coral Isopora palifera, and Symbiodinium C or D types, to assimilate dissolved inorganic carbon (via photosynthesis) and nitrogen (as ammonium). Our results indicate that Symbiodinium types from two clades naturally associated with I. palifera possess different metabolic capabilities. The Symbiodinium C type fixed and passed significantly more carbon and nitrogen to its coral host than the D type. This study provides further insights into the metabolic plasticity among different Symbiodinium types in hospite and strengthens the evidence that the more temperature-tolerant Symbiodinium D type may be less metabolically beneficial for its coral host under non-stressful conditions.

Similar specificities of symbiont uptake by adults and larvae in an anemone model system for coral biology

Reef-building corals depend for much of their energy on photosynthesis by symbiotic dinoflagellate algae (genus Symbiodinium) that live within their gastrodermal cells. However, the cellular mechanisms underpinning this ecologically critical symbiosis, including those governing the specificity of symbiont uptake by the host, remain poorly understood, in part because of the difficulties of working with corals in the laboratory. Here, we used the small symbiotic sea anemone Aiptasia as an experimentally tractable model system to analyze the specificity and timing of symbiosis onset in larval and adult animals under controlled laboratory conditions. Using four clonal, axenic Symbiodinium strains, we found no difference in uptake specificity between larvae (even when very young) and adults. Although both compatible and incompatible algal strains were found within the larval guts, only the former appeared to be internalized by gastrodermal cells, and they (but not incompatible algae) proliferated rapidly within the larvae in the absence of detectable exchange with other larvae. Older larvae showed reduced ingestion of both compatible and incompatible algae, and the addition of food failed to promote the uptake of an incompatible algal strain. Thus, Aiptasia adults and larvae appear to have similar mechanisms for discriminating between compatible and incompatible dinoflagellate types prior to phagocytosis by host gastrodermal cells. Whether a particular algal strain is compatible or incompatible appears to be stable during years of axenic culture in the absence of a host. These studies provide a foundation for future analyses of the mechanisms of symbiont-uptake specificity in this emerging model system.

IMPACT OF BLEACHING STRESS ON THE FUNCTION OF THE OXYGEN EVOLVING COMPLEX OF ZOOXANTHELLAE FROM SCLERACTINIAN CORALS(1).

Global climate change is leading to the rise of ocean temperatures and is triggering mass coral bleaching events on reefs around the world. The expulsion of the symbiotic dinoflagellate algae is believed to occur as a result of damage to the photosynthetic apparatus of these symbionts, although the specific site of initial impact is yet to be conclusively resolved. Here, the sensitivity of the oxygen evolving complex (OEC) to bleaching stress was studied as well as its natural variation between seasons. The artificial electron donor, diphenyl carbazide (DPC), was added to cultured, freshly isolated and expelled (bleaching treatments only) zooxanthellae suspensions. Chl a fluorescence and oxygen production measurements showed that upon addition of DPC, no restoration of diminished photochemical efficiency occurred under control or bleaching conditions. This result was consistent between 12 h and 5 d bleaching treatments on Pocilloporadamicornis, indicating that the OEC is not the primary site of damage, and that zooxanthellae expulsion from the host is a nonselective process with respect to the functioning of the OEC. Further experiments measuring fast induction curves (FICs) revealed that in both summer and winter, the temperature when OEC function was lost occurred between 7°C and 14°C above the sea surface temperature. FIC and oxygen production measurements of P. damicornis during exposure to bleaching stress demonstrated that the thermotolerance of the OEC increased above the temperature of the bleaching treatment over a 4 h period. This finding indicates that the OEC has the capacity to acclimate between seasons and remains functional at temperatures well above bleaching thresholds.

The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.

The ecotoxicological effects of Photosystem II herbicides on corals

The recent discovery of contamination of the tropical marine environment by Photosystem II (PSII) herbicides used in agriculture and antifouling paints has led to concerns regarding the effects on corals and their symbiotic dinoflagellate algae. In reviewing the ecotoxicological studies conducted so far, PSII herbicides appear able to readily penetrate the coral tissues and rapidly (within minutes) reduce the photochemical efficiency of the intracellular algal symbionts. The dinoflagellates appear at least as sensitive to PSII herbicides as other phototrophs tested so far, with photosynthesis being affected at exceptionally low concentrations (i.e. in the ng l −1 range). At these levels and over short exposure periods, the effects can be fully reversible (i.e. when corals are returned to clean seawater) and vary according to type of herbicide; however, when exposed to higher concentrations in the light or over longer exposure periods, it results in a long-term sustained reduction of the photochemical efficiency of the algae (symptomatic of chronic photoinhibition). This can result in the dissociation of the symbiosis (bleaching) which is a common but nevertheless significant sub lethal stress response requiring many months to recover from. It is argued that the reliance of corals on an endosymbiotic photoautotrophic energy source, together with predilection for the symbiosis to dissociate when photosynthesis of the algae is affected, renders coral particularly susceptible to changes in environmental conditions—and especially phytotoxins such as PSII herbicides.

The effects of Produced Formation Water (PFW) on coral and isolated symbiotic dinoflagellates of coral

There is concern of the effects of Produced Formation Water (PFW, an effluent of the offshore oil and gas industry) on temperate/tropical marine organisms of the North West Shelf (NWS) of Australia. Little is known of the effects of PFW on tropical marine organisms, especially keystone species. Exposing the coral Plesiastrea versipora to a range (3–50% v/v) of PFW from Harriet A oil platform resulted in a reduction in photochemical efficiency of the symbiotic dinoflagellate algae in hospite (in the coral tissues), assessed as a decrease in the ratio of variable fluorescence (Fv) to maximal fluorescence (Fm) measured using chlorophyll fluorescence techniques. Significant differences were noted at PFW concentrations &gt;12.5% (v/v). In corals where Fv/Fm was significantly lowered by PFW exposure, significant discolouration of the tissues occurred in a subsequent 4-day observation period. The discolouration (coral bleaching) was caused by a loss of the symbiotic dinoflagellates from the tissues, a known sublethal stress response of corals. PFW caused a significant decrease in Fv/Fm in symbiotic dinoflagellates freshly isolated from the coral Heliofungia actiniformis at 6.25% PFW, slightly lower than the studies in hospite. Corals exposed to lower PFW concentrations (range 0.1%–10% PFW v/v) for longer periods (8 days) showed no decrease in Fv/Fm, discolouration, loss of symbiotic dinoflagellates or changes in gross photosynthesis or respiration (measured using O2 exchange techniques). The study demonstrates minor toxicity of PFW from Harriet A oil platform to corals and their symbiotic algae.

Nitric Oxide and Cnidarian-Dinoflagellate Symbioses: Pieces of a Puzzle1

Abstract The presence of nitric oxide synthase (NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida and in its symbiotic dinoflagellate algae, Symbiodinium bermudense. Enzyme activity was assayed by measuring the conversion of arginine to citrulline. Biochemical characterization of NOS from Aiptasia was characterized with respect to cellular localization, substrate and cofactor requirements, inhibitors, and kinetics. In response to acute temperature shock, anemones retracted their tentacles. Animals subjected to such stress had lower NOS activities than did controls. Treatment with NOS inhibitors caused tentacular retraction, while treatment with the NOS substrate L-arginine inhibited this response to stress, as did treatment with NO donors. These results provide a preliminary biochemical characterization of, and suggest a functional significance for, NOS activity in anthozoan-algal symbiotic assemblages.

Inhibition of algal photosynthesis by a symbiotic coral

When symbiotic dinoflagellate algae (Symbiodinium sp.), isolated from the scleractinian coral Plesiastrea versipora were incubated in homogenized host tissue, carbon fixation was usually lower than in seawater only (43 cases of of 54), and this inhibition occurred throughout the year. Algae incubated in homogenized host tissue fixed a mean of 78.84% ± 22.6% of the amount fixed by algae in seawater (n = 54, range 33–142%). The difference between rates of fixation in seawater (mean ± SD of 61.300 ± 22.71 nmol C/105 cells) and in homogenates (mean ± SD of 47.377 ± 21.42 nmol C/105 cells) in individual experiments, was highly significant (p<0.0001, paired t-test). Homogenized host tissue from populations of P. versipora separated by 4000 km inhibited photosynthesis in algae from both their own and the distant coral population. Inhibition of photosynthesis was not observed when isolated algae from the anemone, Aiptasia pulchella were incubated in homogenate from P. versipora. An inhibitor of photosynthesis was isolated from a low molecular weight fraction of homogenized host tissue of P.versipora by cation-exchange chromatography and further purified by HPLC. This is the first natural inhibitor of photosynthesis that has been found in an invertebrate with symbiotic algae.

Low molecular-weight factor from Plesiastrea versipora (Scleractinia) that modifies release and glycerol metabolism of isolated symbiotic algae

When symbiotic dinoflagellate algae (Symbiodinium sp., isolated from the coral Plesiastrea versipora) were incubated with NaH14CO3 in the light in seawater, they released 22.69±9.16 nmol carbon/106 algae. Release of photosynthetically fixed carbon was stimulated more than six-fold for algae incubated in host-tissue homogenate (148.54±97.03 nmol C/106 algae) and more than four-fold (102.00±49.16 nmol C/106 algae) for algae incubated in a low molecular weight fraction (≤1 000 M r ) prepared from host homogenate. Soluble released 14C-labelled products, as determined by chromatography and autoradiography, were the same when algae were incubated in either host homogenate or the low molecular weight fraction. After 4 h incubation in the light (300 mol photons m−2 s−1),␣intracellular␣glycerol increased in algae incubated with the low molecular weight fraction (an increase of 0.39 to␣0.67 nmol glycerol/106 algae) compared with little or no increase in algae incubated in seawater (0 to 0.12 nmol glycerol/106 algae). Partial inhibition of triglyceride synthesis (up to 51%) was also observed when algae were incubated in the low molecular weight fraction. All these effects are the same as those observed when algae were incubated in host homogenate. These data indicate that the “host release-factor” activity of P.␣versipora is a compound of low molecular weight.

Effects of host-tissue homogenate of the scleractinian coral Plesiastrea versipora on glycerol metabolism in isolated symbiotic dinoflagellates

Symbiotic dinoflagellate algae (Symbiodinium sp.) isolated from the scleractinian coral Plesiastrea versipora and incubated in homogenized host tissue released 4 to 7 times as much glycerol (14 to 46 nmol glycerol/106 algae) as those incubated in seawater (3 to 6 nmol glycerol/106 algae) after 4 h incubation in the light. During this period, no release of triglycerides was detected. Intracellular glycerol increased 2- to 3-fold in algae incubated in host homogenate, but remained unchanged in algae incubated in seawater at a concentration of 0.82 ± 0.47 nmol glycerol/106 algae. In each incubation condition, intracellular triglyceride levels increased. However, in algae incubated in host homogenate, the intracellular levels of triglycerides reached only about 75% of the amount reached in algae incubated in seawater (max. 18.55 ± 2.40 nmol glycerol/106 cells). Host homogenate did not stimulate the release of glycerol from algae during dark incubation. These data show that the glycerol released by algae incubated in host-tissue homogenate was derived from increased synthesis of glycerol or from diversion of some glycerol or other photosynthetic intermediates from incorporation into algal triglyceride stores, and did not come from existing stores.