The Establishment of the Hydra-Chlorella Symbiosis: Recognition of Potential Algal Symbionts

The epithelial cells lining the gastric cavity of the freshwater hydra, Hydra viridis, harbor unicellular algal symbionts of the genus Cblorella. It has long been known that these hydra cells can readily phagocytose algal cells and will sequester those algae that have the potential to form a symbiotic association. In this paper the evidence is discussed for when and how recognition of potential symbionts by hydra cells occurs, i.e. during phagocytosis or during the subsequent intracellular events leading to sequestration of algal symbionts.     

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     

A review of some physiological and evolutionary aspects of body size and bud size of Hydra

Green hydra with endosymbionts are smaller than brown asymbiotic ones. Regeneration experiments, mitotic index studies on algal and hydra tissue, and evidence for consumption and expulsion of algae are reviewed and it is suggested that larger green hydra have more difficulty controlling algal increase than smaller ones and that hydra have an upper size limit for maintenance of stable symbioses. A mathematical model is discussed which starts with simple physiological assumptions about hydra and generates field testable conclusions about how body and bud size, and reproductive rates depend on food particle size, quantity and temporal distribution. Unlike most analytic ecological-evolutionary models, this one integrates physiology, ecology and evolution without needing simplifying assumptions.     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

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.     

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.     

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.     

Amino acids as a nitrogen source for Chlorella symbiotic with green hydra

Supply of amino acids may be important in controlling cell division of Chlorella symbiotic with green hydra. Freshly isolated symbionts display characteristics of N-limited algae, and low pH in perialgal vacuoles and high levels of host glutamine synthetase (GS) limit uptake of ammonium. Movement of tritiated amino acids from host to algal pools suggests that symbiotic algae utilize amino acids derived from host digestion of prey. Amounts are significant in relation to host and algal amino acids pools. During host starvation, glutamine produced by host GS may be important as a nitrogen supply to the algae, which take up this amino acid at high rates at low pH.     

Expulsion of Symbiotic Algae during Feeding by the Green Hydra – a Mechanism for Regulating Symbiont Density?

Background

Algal-cnidarian symbiosis is one of the main factors contributing to the success of cnidarians, and is crucial for the maintenance of coral reefs. While loss of the symbionts (such as in coral bleaching) may cause the death of the cnidarian host, over-proliferation of the algae may also harm the host. Thus, there is a need for the host to regulate the population density of its symbionts. In the green hydra, Chlorohydra viridissima, the density of symbiotic algae may be controlled through host modulation of the algal cell cycle. Alternatively, Chlorohydra may actively expel their endosymbionts, although this phenomenon has only been observed under experimentally contrived stress conditions.

Principal Findings

We show, using light and electron microscopy, that Chlorohydra actively expel endosymbiotic algal cells during predatory feeding on Artemia. This expulsion occurs as part of the apocrine mode of secretion from the endodermal digestive cells, but may also occur via an independent exocytotic mechanism.

Significance

Our results demonstrate, for the first time, active expulsion of endosymbiotic algae from cnidarians under natural conditions. We suggest this phenomenon may represent a mechanism whereby cnidarians can expel excess symbiotic algae when an alternative form of nutrition is available in the form of prey.     

Molecular approaches to diagnosing nutritional physiology in harmful algae: Implications for studying the effects of eutrophication

Every year harmful algal blooms (HABs) cause serious impacts to local economies, coastal ecosystems, and human health on a global scale. It is well known that nutrient availability can influence important aspects of harmful algae biology and ecology, such as growth, toxin production, and life cycle stage, as well as bloom initiation, persistence and decline. Increases in the rate of supply of organic matter to ecosystems (eutrophication) carries many possible ramifications to coastal systems, including the potential for nutrient enrichment and the potential for stimulation of harmful algal blooms. Traditional studies on algal nutrition typically use either cultured isolates or community level assays, to examine nutrient uptake, nutrient preference, elemental composition, and other metrics of a species’ response to nutrients. In the last decade, technological advances have led to a great increase in the number of sequences available for critical harmful species. This, in turn, has led to new insights with regards to algal nutrition, and these advances highlight the promise of molecular technologies, and genomic approaches, to improving our understanding of algal nutrient acquisition and nutritional physiological ecology, in both cultures and field populations. With these developments increased monitoring of nutritional physiology in field populations of harmful algae will allow us to better discriminate how eutrophication impacts these groups.     

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.     

Territorial behavior of threespot damselfish (Eupomacentrus planifrons) increases reef algal biomass and productivity

Synopsis Algal growth and damselfish (Eupomacentrus planifrons) territories were studied in two reef habitats at Discovery Bay, Jamaica. Damselfish territories were contiguous in the reef flat (0 to 2.5 m), where the algal composition and biomass varied from territory to territory. In contrast, on the lower reef terrace (22 m), damselfish territories were often spatially segregated. While the algal composition of the territories was more uniform on the reef terrace, the total algal biomass was lower than in the territories on the reef flat. Damselfish are largely herbivorous, and they defend their territories against most intruding fish, including a number of herbivorous species. Areas of the reef terrace outside of damselfish territories were heavily grazed by herbivorous fishes and contained only small quantities of non-crustose algae.The reef terrace territories were characterized by a multispecific turf of algae (greens, blue-greens, and reds) covering the Acropora cervicornis framework and by the leafy, brown alga, Lobophora variegata. A rapid reduction in the biomass of brown algae and filamentous algae was noted when damselfish were permanently removed from their territories. Only calcified, encrusting algae — plants apparently somewhat undesirable as fish food sources — would be common on the terrace zone of this reef if damselfish territories were absent. Damselfish territoriality may significantly influence the dynamics of some reefs by increasing the biomass of the algal turf thereby increasing; reef productivity. Since blue-green algae, potential nitrogen fixers, occur in these algal turfs, the fish may also be indirectly affecting reef nutrition.     

Growth and survival of protozoa isolated from a tannery effluent

Industrial effluent from a tannery was used for the growth of algae in a medium containing various inorganic salts. Growth of algal cells became visible after 7 d. Two species of protozoa were observed to proliferate in the algal culture containing no organic supplement in the medium. The culture was kept bacteria-free by the use of antibiotics and was perpetuated for at least 150 d with no decline in the protozoan population. Efficient growth of protozoa in a culture of algae elucidated new modes of nutrition in protozoa. Cr(VI) was added to the medium to check the resistance of algae and protozoa against this heavy metal. Protozoa showed different degrees of resistance. The results indicate the importance of algae and protozoa in the process of bioremediation.     

Localization and evolution of septins in algae

Septins are a group of GTP‐binding proteins that are multi‐functional, with a well‐known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog‐possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.     

Experimental identification and in silico prediction of bacterivory in green algae

While algal phago-mixotrophs play a major role in aquatic microbial food webs, their diversity remains poorly understood. Recent studies have indicated several species of prasinophytes, early diverging green algae, to be able to consume bacteria for nutrition. To further explore the occurrence of phago-mixotrophy in green algae, we conducted feeding experiments with live fluorescently labeled bacteria stained with CellTracker Green CMFDA, heat-killed bacteria stained with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), and magnetic beads. Feeding was detected via microscopy and/or flow cytometry in five strains of prasinophytes when provided with live bacteria: Pterosperma cristatum NIES626, Pyramimonas parkeae CCMP726, Pyramimonas parkeae NIES254, Nephroselmis pyriformis RCC618, and Dolichomastix tenuilepis CCMP3274. No feeding was detected when heat-killed bacteria or magnetic beads were provided, suggesting a strong preference for live prey in the strains tested. In parallel to experimental assays, green algal bacterivory was investigated using a gene-based prediction model. The predictions agreed with the experimental results and suggested bacterivory potential in additional green algae. Our observations underline the likelihood of widespread occurrence of phago-mixotrophy among green algae, while additionally highlighting potential biases introduced when using prey proxy to evaluate bacterial ingestion by algal cells.Subject terms: Microbial ecology, Cellular microbiology     

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.     

Benthic macroalgae as a dispersal mechanism for fauna: influence of a marine tumbleweed

Effective dispersal is problematic for benthic organisms without planktonic larvae; rafting and vertical migrations are mechanisms that can potentially be employed by such fauna, but these strategies entail considerable predation risk as well as other disadvantages. Unattached, but non-floating, “drift” algae harbor large numbers of fauna and may serve as an alternative dispersal mechanism in some systems. This paper reports field manipulations in Florida Bay, Florida, USA designed to determine (1) if such algae can disperse benthic animals, and (2) if dispersal efficiency varies as a function of two common substrata types: seagrass and bare sediment. A live immersion stain was used to mark faunal associates of Laurencia spp. algal clumps in situ. The fidelity of molluscs, decapods, ophiuroids, and fishes to stationary algal clumps was then compared with the fidelity of these animals to clumps that were forced to tumble over a given distance with a blower apparatus; these experiments were performed over both sand and seagrass substrata. Measurements of frequency, spatial extent, and rate of algal drift were made to aid in assessing the potential importance of benthic algae as a dispersal mechanism.

Algal clumps often rolled in a manner similar to that of terrestrial tumbleweeds; mark-recapture work showed that algal clumps can move up to 0.5 km/day and that algal drift is a frequent phenomenon. The algal masses were effective transporters of benthic fauna, including mobile shrimps and fishes; dispersal was more efficient over sand than over seagrass. Dispersal of fauna via this mobile habitat should entail lower risk than other adult dispersal stratagems such as vertical migration or rafting; this mechanism would be most advantageous for brooding species or those with limited planktonic phases. Differential fidelity to clumps tumbling across seagrass versus sand suggests that the algae could facilitate exchange of fauna between isolated seagrass patches.     

Limitations of Using Cultured Algae to Study Cnidarian‐Algal Symbioses and Suggestions for Future Studies

Much of our understanding of the cellular mechanisms underlying cnidarian‐algal symbiosis comes from studying the biological differences between the partners when they are engaged in symbiosis and when they are isolated from one another. When comparing the in hospite and ex hospite states in Symbiodiniaceae, the in hospite state is represented by algae sampled from hosts, and the ex hospite state is commonly represented by cultured algae. The use of cultured algae in this comparison may introduce nutrition as a confounding variable because, while hosts are kept in nutrient‐depleted conditions, culture media is nutrient rich and designed to facilitate algal growth. In this perspective, we reexamine how nutrition may be a confounding variable in studies that compare the biology of Symbiodiniaceae in hospite and in culture. We also suggest several innovations in experimental design to strengthen the comparison of the two lifestyles, including the adoption of nutritional controls, alternatives to culture for the representation of Symbiodiniaceae ex hospite, and the adoption of several proteomic approaches to find novel Symbiodiniaceae genes important for symbiosis.     

The ins and outs of algal metal transport

Metal transporters are a central component in the interaction of algae with their environment. They represent the first line of defense to cellular perturbations in metal concentration, and by analyzing algal metal transporter repertoires, we gain insight into a fundamental aspect of algal biology. The ability of individual algae to thrive in environments with unique geochemistry, compared to non-algal species commonly used as reference organisms for metal homeostasis, provides an opportunity to broaden our understanding of biological metal requirements, preferences and trafficking. Chlamydomonas reinhardtii is the best developed reference organism for the study of algal biology, especially with respect to metal metabolism; however, the diversity of algal niches necessitates a comparative genomic analysis of all sequenced algal genomes. A comparison between known and putative proteins in animals, plants, fungi and algae using protein similarity networks has revealed the presence of novel metal metabolism components in Chlamydomonas including new iron and copper transporters. This analysis also supports the concept that, in terms of metal metabolism, algae from similar niches are more related to one another than to algae from the same phylogenetic clade. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Nutritional value of algae: a critical control on the stability of Daphnia-algal systems

In Daphnia–algal systems, the effect of nutrient enrichmenton stability is an important ecological issue. Here I considera system of Daphnia and two potential prey; one prey termedprimary algae, which are preferentially consumed, and the othersecondary algae, which yield less nutrition and are more resistantto the grazer. The presence of secondary algae is a key to thestability, but their nutritional value has not been clearlydefined in the previous theory and the actual value varies.Here I use a simple mathematical model defining explicitly thenutritional values of algae and examine the stability of thesystems as a function of phosphorus enrichment. Whether or notall three species can stably coexist depended on the combinationof the algal species used for simulation. In systems where allthe species coexist in a stable manner, in which enrichmentdoes not necessarily lead to destabilization, there is alwaysa critical nutritional value of the secondary algae. Empiricalwork supports the possibility that the unknown nutritional valueof secondary algae takes a value close to the critical one.Furthermore, at the critical nutritional value, the populationresponse in the systems to enrichment is consistent with theobserved trend in natural systems. This suggests that Daphnia–algalsystems in nature can maintain stability in the face of enrichment,without requiring specific assumptions such as spatial heterogeneity.