Delay in migration of symbiotic algae in Hydra viridis by inhibitors of microtubule protein polymerization

An English strain of the fresh water symbiotic coelenterate Hydra viridis was experimentally "bleached" of its Chlorella algae and maintained indefinitely by feeding. The algal symbiosis could be re-established by injecting other symbiotic algae into aposymbionts. Although algal uptake and recognition were not affected by microtubule protein polymerization inhibitors, these compounds i.e., podophyllotoxin, beta-peltatin and vinblastine had delaying effects on the migration of the algae through the host digestive cells. Picropodophyllotoxin did not delay migration. The rates, the reversibility and the sensitivity of algal migration to low concentrations of drugs known to bind tubulin suggests the symbionts migrate somehow via labile polymerization of host hydra tubulin into microtubules.     

Glucose uptake by symbiotic Chlorella in the green-hydra symbiosis

There is a correlation between the ability of symbiotic Chlorella algae to take up glucose and their survival in green hydra grown in continuous darkness. Although normal symbionts of European green hydra, which persist at a stable level in dark-grown animals, possessed no detectable constitutive ability to take up glucose when grown in light, uptake was induced after incubation in a medium containing glucose. Further, symbionts isolated from hydra grown in darkness for two weeks had acquired a constitutive uptake ability. Neither NC64A nor 3N813A strains of algae, in artificial symbiosis with hydra, persisted in dark-grown animals, and they showed little or no uptake ability, although in culture NC64A possessed both constitutive and inducible glucose-uptake mechanisms. In contrast, mitotic indices in all three types of algae in symbiosis with hydra increased after host feeding, indicating that the factor which stimulates algal cell division is not identical to the substrate utilised during heterotrophic growth.Abbreviations E/E normal Hydra-Chlorella symbiosis - E/NC, E/3N artificial symbioses between Hydra and Chlorella strains NC64A and 3N813A, respectively - 3-OMG 3-O-methyl-D-glucose - SDS sodium dodecyl sulfate     

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.     

New observations on green hydra symbiosis

New observations on green hydra symbiosis are described. Herbicide norflurazon was chosen as a "trigger" for analysis of these observations. Green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoeptihelial cells it contains individuals of Chlorella vulgaris Beij. (KESSLER & HUSS 1992). Ultrastructural changes were observed by means of TEM. The newly described morphological features of green hydra symbiosis included a widening of the perialgal space, missing symbiosomes and joining of the existing perialgal spaces. Also, on the basis of the newly described mechanisms, the recovery of green hydra after a period of intoxication was explained. The final result of the disturbed symbiosis between hydra and algae was the reassembly of the endosymbiosis in surviving individuals.     

The morphology of green hydra endosymbionts as influenced by host strain and host environment.

The ultrastructure of Chlorella-like algal endosymbionts from the Florida and English strains of green hydra was compared under different host feeding and photoperiodic regimes. Under standard conditions (host fed daily, 12-h photoperiod) the algae from the 2 strains exhibited considerable differences. The English symbionts had a pyrenoid, compact chloroplast membranes and vesiculated polyphosphate bodies. By comparison, Florida symbionts lacked a pyrenoid, had chloroplasts with less compact membranes and exhibited spherical polyphosphate bodies. When maintained in the dark, algae from English hydra lost their pyrenoids, showed great compaction of the chloroplast and developed large, shield-shaped, electron-dense bodies. In contrast, algae from Florida hosts did not exhibit gross ultrastructural modification. Reciprocal cross-transfers of symbionts were made by placing Florida algae in English aposymbiotic (algal-free) hosts and vice versa. After residence in Florida hosts, English symbionts appeared to undergo ultrastructural modifications resulting in a morphology indistinguishable from the native Florida symbionts. Florida algae showed no modifications resulting from residence in English hosts. It thus appears that the English symbiont has great morphological plasticity, as its structure is greatly modified depending upon the host in which it resides and the conditions under which the host is maintained. The results of these studies are discussed and compared with published accounts of free-living Chlorella and with reports dealing with other Chlorella symbionts.     

Symbiosis between hydra and chlorella: Molecular phylogenetic analysis and experimental study provide insight into its origin and evolution

Although many physiological studies have been reported on the symbiosis between hydra and green algae, very little information from a molecular phylogenetic aspect of symbiosis is available. In order to understand the origin and evolution of symbiosis between the two organisms, we compared the phylogenetic relationships among symbiotic green algae with the phylogenetic relationships among host hydra strains. To do so, we reconstructed molecular phylogenetic trees of several strains of symbiotic chlorella harbored in the endodermal epithelial cells of viridissima group hydra strains and investigated their congruence with the molecular phylogenetic trees of the host hydra strains. To examine the species specificity between the host and the symbiont with respect to the genetic distance, we also tried to introduce chlorella strains into two aposymbiotic strains of viridissima group hydra in which symbiotic chlorella had been eliminated in advance. We discussed the origin and history of symbiosis between hydra and green algae based on the analysis.     

Value of the Hydra model system for studying symbiosis

Green Hydra is used as a classical example for explaining symbiosis in schools as well as an excellent research model. Indeed the cosmopolitan green Hydra (Hydra viridissima) provides a potent experimental framework to investigate the symbiotic relationships between a complex eumetazoan organism and a unicellular photoautotrophic green algae named Chlorella. Chlorella populates a single somatic cell type, the gastrodermal myoepithelial cells (also named digestive cells) and the oocyte at the time of sexual reproduction. This symbiotic relationship is stable, well-determined and provides biological advantages to the algal symbionts, but also to green Hydra over the related non-symbiotic Hydra i.e. brown hydra. These advantages likely result from the bidirectional flow of metabolites between the host and the symbiont. Moreover genetic flow through horizontal gene transfer might also participate in the establishment of these selective advantages. However, these relationships between the host and the symbionts may be more complex. Thus, Jolley and Smith showed that the reproductive rate of the algae increases dramatically outside of Hydra cells, although this endosymbiont isolation is debated. Recently it became possible to keep different species of endosymbionts isolated from green Hydra in stable and permanent cultures and compare them to free-living Chlorella species. Future studies testing metabolic relationships and genetic flow should help elucidate the mechanisms that support the maintenance of symbiosis in a eumetazoan species.     

Orthophosphate influx and efflux rates of Chlorella fusca measured in a continuous turbidostat culture with 32P under various conditions

The main objective of the experiments with Chlorella fusca strain 211-8b was to measure, with adequate time resolution, the unidirectional influx rates of phosphate into non-phosphate-starved algae under different steady state conditions (light, temperature, 3-phosphoglycerate influence) or following the addition of several photosynthesis and phosphate transport inhibitors (phenylmercuric acetate, p-chloromercuribenzoate, arsenate). the algae were cultivated in a phosphate rich medium in a continuous turbidostat culture. The phosphate exchange experiments with carrier-free ³²PO ₄ ^3- were performed directly in the continuous culture. The sampling intervals after the tracer addition were 15 s.For a continuous steady state culture grown in the light (25° C) the unidirectional influx rate measured with ³²P is 260 times higher than the net uptake rate (=influx minus efflux rate) calculated from the mass balance using the data of this culture. In all experiments, except the control experiment with trichloroacetic acid killed cells, the specific activity of the intracellular inorganic orthophosphate compartment oscillates around a constant mean value which never reaches the specific activity of the nutrient medium within the duration of the short-term experiments (7.5 min). The inhibitors strongly affect the characteristics of the oscillations. The unidirectional influx rates are constant. Oscillating flushing rates with unlabelled phosphate from a storage compartment have been postulates to explain the oscillations. Oscillating rates from the individual cells are apparently synchronized by an unknown mechanism.     

Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra

Symbiotic associations are of a wide significance in evolution and biodiversity. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells, it harbors individuals of unicellular green algae. Morphological characteristics of isolated algae determined by light and electron microscopy are presented. Cytological morphometric parameters (cell area, cell radius, chloroplast area) of isolated algae from green hydra (Cx), as well as from reference species Chlorella kessleri (Ck) and Chlorella vulgaris (Cv), revealed similarity between the isolated endosymbiont and C. kessleri. Isoenzyme patterns of esterase (EST), peroxidase (POX), and catalase (CAT) were used for the investigation of genetic variability in endosymbiotic algae isolated from green hydra. Out of 14 EST isoenzymes observed in Cx species, 9 were expressed in the Cx sample. Results of the EST isoenzyme analysis indicated a higher degree of similarity between Cx and Cv than between Cx and Ck. Due to much higher heterogeneity, EST isoenzymes seem to be more suitable genetic markers for identification of different Chlorella species than CAT and POX isoenzymes. Results obtained suggest that symbiogenesis in green hydra has probably not been terminated yet.     

Comparative evaluation of the vitamin composition of unicellular algae and higher plants grown under artificial conditions

The vitamin composition of representatives of green (Chlorella vulgaris, Platimonas viridis), blue-green (Synechococcus elongatus, Coccopedia, Spirulina platensis, Cyanidium caldarium), red (Porphyridium cruentum) unicellular algae and higher plants (wheat, chufa, beet, carrot, turnip, radish, cucumber, dill, Welsh onion, potato) grown under artificial conditions was examined. The content of B complex vitamins (thiamine, riboflavine, nicotinic and folic acids), ascorbic acid and carotene was measured. Among the algae studied Chlorella vulgaris and Spirulina platensis showed the highest vitamin activity. The red alga Porphyridium cruentum contained the lowest quantity of thiamine, riboflavine and carotene and larger amounts of nicotinic acid. Comparison of the content of vitamins C, B1, B2, PP, folic acid and carotene in unicellular algae and higher plants, that are natural and traditional sources of the vitamins, demonstrated that the above green and blue-green algae contain greater than higher plants amounts of thiamine, riboflavine, folic acid and carotene, when calculated per g dry matter. All algae, except for Platimonas viridis and Cyanidium caldarium, are superior to beet and carrot in their content of ascorbic acid and inferior to green vegetables (radish, cabbage, dill and Welsh onion) in that parameter.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

小球藻藻菌共生体系在产油方面的特性

【目的】研究人工构建藻菌共生体系在产油方面的特性。【方法】从BG11培养基中分离、筛选出无菌小球藻,通过人工共培养方法构建了藻菌共生体系,探讨了共生体系中小球藻的生长及产油特性。【结果】相比无菌小球藻,藻菌共生体系对于藻的生长、油脂积累以及产生生物柴油的脂肪酸组分方面都有明显的促进作用,其中细菌(Stenotrophomonas maltophilia)和小球藻构建的共生体系效果最好,小球藻生物量提高了9%,油脂含量提高了36.3%,C18-1的含量提高了259.2%。【结论】进一步说明人工共培养方法构建藻菌共生体系能够提高微藻生物柴油的质量,具有很好的利用价值。     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

Transmission of symbiotic algae through sexual reproduction in hydra: movement of algae into the oocyte

The histological pathway by which intracellular symbiotic Chlorella move into the developing oocytes of hydra was investigated at the ultrastructural level. Algae move from within the digestive cells of the endoderm to within the oocytes of the ectoderm in a three-step process. First, the algae are released by digestive cells into the mesolamella (basement membrane). Second, the algae move as individual cells into the adjacent intercellular spaces of the ectoderm. Third, they are taken up by the oocyte by phagocytosis. This transfer occurs only in the central regions of the ovary, and only after oocytes have reached an advanced stage. Normally the mesolamella is separated from the ectodermal interstitial spaces by a layer of epitheliomuscular cell muscle processes. This layer degenerates in the region where algae will move into the ectoderm. This study shows that algae move as individual cells and not intracellularly within processes of the epithelial cells.     

Uptake and accumulation of exogenous docosahexaenoic acid by Chlorella

Tuna oil or its hydrolysate was added to a culture of Chlorella for its nutritional fortification as a feed for rotifer. Exogenous docosahexaenoic acid (DHA) in its free form was taken up by the cells of Chlorella vulgaris strain K-22 and by other strains, but tuna oil was not taken up by the cells. Accumulated DHA was found by electron microscopy in the cells in oil droplets. All strains of Chlorella used in these experiments took up exogenous DHA into the cells. It seems that the structure of the cell wall did not affect the uptake of DHA into the Chlorella cells.     

Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis

Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatase nor ferritin was delivered via lysosomes into phagosomes containing live symbionts, whereas these lysosomal markers were found in 50% of the vacuoles containing heat-killed symbionts 1 h after phagocytosis. Treatment of symbiotic algae before phagocytosis with polycationic polypeptides abolishes algal persistence and perturbs the ability of these algae to control the release of photosynthate in vitro. Similarly, inhibition of photosynthesis and hence of the release of photosynthetic products as a result of prolonged darkness and 3-(3,4- dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment also abolishes persistence. Symbiotic algae are not only protected from host digestive attack but are also selectively transported within host cells, moving from the apical site of phagocytosis to a basal position of permanent residence. This process too is disrupted by polycationic polypeptides, DCMU and darkness. Both algal persistence and transport may, therefore, be a function of the release of products from living, photosynthesizing symbionts. Vinblastine treatment of host animals blocked the movement of algae within host cells but did not perturb algal persistence: algal persistence and the transport of algae may be initiated by the same signal, but they are not interdependent processes.     

Metabolism of Urea by Chlorella vulgaris

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Endosymbiotic bacteria associated with the intracellular green algae ofHydra viridis

Gram-negative bacteria 4.5–5.5 μm in length and 1.2 μm in diameter are found in gastrodermal cells of three stains of freshwater green hydras,Hydra viridis (Ohio and Carolina from North America, Jubilee strain from England). They are motile via single polar flagella. They were detected in live animals, Jensen stained material, and electron micrographic sections. Bacteria lose motility quickly upon release from hydra cells. Green hydras harbor strain-specific numbers of chlorellae in these cells. Other tissue types lack algae. The chlorella-hydra symbiosis can be disassociated and the partners grown separately; transfer of photosynthate from algae to hydra occurs. Here we report the presence of endocellular bacterial vesicles specifically associated with cells that contain the symbiotic chlorellae. No cells that contained algae and lacked bacteria were seen. Vesicles, especially conspicuous in sexually mature green hydras, are probably produced upon extrusion from the cell. They contain either algae and bacteria or bacteria alone and are often expelled to the surrounding medium via the coelenteron. Bacteria are absent in nerve cells, interstitial cells, nematocysts, mucous cells, sperm, and probably in most of the other cell types that lack algae. They are present in at least one cell type that lacked algae: the columnar ovarian cells. Bacteria were lost in “bleached” hydras, those induced to lose their algae by high intensity light in a solution of DCMU, a standard inhibitor of photosynthesis. They were absent in a fourth strain of green hydra (Connecticut Valley,H. viridis) and inH. fusca andH. littoralis, two freshwater nonsymbiotic hydras. All of the hydra lacking bacteria contain conspicuous lipid droplets in their cells. The presence of large numbers of bacteria has implications for interpretations of metabolic exchange between host and algal symbionts and for extrapolation of metabolic data from strain to strain ofH. viridis.