Two aeromonads: Growth of symbionts fromHydra viridis

A large population of bacteria resides in the gastrodermal and ovarian tissue of the freshwater green coelenterateHydra viridis (Ohio, Jubilee, and Carolina strains). The intracellular bacteria are strongly correlated with the presence of symbiotic chlorellae in the animal cells. The bacteria accompany the chlorellae when they are expelled in vesicles during stages of sexual maturation. Two isolates of these bacteria were taken from ruptured bacterial-algal vesicles flushed out of the enteron of surface-sterilzed hydras. They were cultured on proteose peptone. Both were identified asAeromonas punctata, on the basis of over forty traits. These large, Gram-negative rods differ very little from published characteristics ofA. punctata subsp.punctata. UnlikeA. punctata subsp.punctata, the hydra symbionts grew on KCN broth and lacked the lysine decarboxylase reaction. We identified the two isolates as members of the same new strain ofAeromonas punctata symbiotic inHydra viridis. A different aeromonad could not be isolated from vesicles flushed out of surface-sterilized hydras, but it appeared along with the first aeromonad on plates from which smears of the holdfast and hypostome region of ethanol-rinsed hydra were made. This second orange-pigmented bacterium in a member of the holdfast microbial community associated with hydra and hydra eggs. It differed fromAeromonas hydrophila subsp.anaerogenes in only 5 out of over 40 traits tested. All differences were losses of metabolic activities. Both hydra-associatedA. hydrophila andA. punctata, which are easily grown, and yet form regular and natural associations with the greenHydra viridis, may prove useful for understanding metabolic relationships in micro-organisms adapted for symbiotic associations.     

Microscopical aspects on symbiosis of Spongilla lacustris (Porifera,Spongillidae) and green algae

Summary When growing in the sunlight, some specimens of Spongilla lacustris are coloured green due to the presence of symbiotic unicellular chlorellae. The algae live inside most sponge cells. The chlorellae were extracted from green sponges, cultivated, added to algae-free sponges and fixed after different incubation times. In this way the uptake of the algae, their distribution and their final whereabouts in the mesenchymatic cells could be followed by in vivo microscopy, phase-contrast microscopy and electron microscopy. A few minutes after addition, the chlorellae can be found inside the choanocyte chambers. Here they are taken up by the cell bodies and collars of the choanocytes. Pinacocytes are also involved in the uptake. The distribution of algae results from a specific transmission from the donor cell to the receiver cell. The chlorellae are not released from their host vacuoles until they are extensively enclosed by the cell taking them up. Six hours after addition, all sponge cells contain algae except granulocytes, microscleroblasts, the pinacocytes of the peripheral rim region and those of the pinacoderm. The chlorellae are able to divide inside the sponge cells.Abbreviations StM Stereo-microscopical photograph - PhC Phase-contrast microscopical photograph - EM Electron microscopical photograph     

The endosymbiotic unit ofStentor polymorphus andChlorella sp. morphological and physiological studies

Summary The greenStentor polymorphus harbours unicellular coccoid chlorophycean algae. They are located in food vacuoles, where they show various states of digestion, as well as in individual so-called perialgal vacuoles. According to their characteristic morphological properties the algae belong to the genusChlorella. They can be isolated from the ciliate and cultivated in mass cultures in a sterile defined inorganic medium supplemented with vitamins B₁ and B₁₂. The algae have no secondary carotenoids and excrete maltose by a pH-dependent mechanism. They thus show a conspicuous physiological similarity to the symbiotic chlorellae ofParamecium bursaria andHydra viridis, which also excrete maltose.A comparison of the properties of the chlorellae isolated fromStentor polymorphus and of the intactStentor polymorphus-Chlorella unit with the characteristic features of symbiotic chlorellae and with endosymbiotic systems containingChlorella sp. in general, lead to the conclusion that the greenStentor polymorphus is also a true endosymbiotic system.     

Interaction of a living system with an electromagnetic field

Irradiation of the inoculum with 50 Hz frequency electromagnetic field for 3 h practically did not influence the growth of suspension culture of one-celled green algae Dunaliella tertiolecta and Tetraselmis viridis. The irradiation for 4 h led to approximately 30% reduction of growth. After 6-hour irradiation the growth of Dunaliella tertiolecta was reduced by 50% and the growth of Tetraselmis viridis was reduced by 70%. The irradiation of hydras for 3 h showed no visible effect. The irradiation for 4 h stimulated a little the growth and regeneration of injured hydras. The effect of 24 h irradiation resulted in the shortening of lifetime of uninjured and injured hydras. The effect of regeneration was not observed.     

The relationship between size,budding rate,and growth efficiency in three species of hydra

Three species of the fresh water carnivore hydra, H. littoralis, H. pseudoligactis, and C. viridissima present a graduation in size with the first species the largest and albino Chlorohydra the smallest. When presented with a daily overabundance of food (artemia), considerable variation in food intake and gross efficiency of growth (proportion of food energy consumed that is turned into new protoplasm or buds) existed among the species. The degree of association between size of species and food intake was highly significant. However, budding efficiency among the species was found to be independent of food intake (when the effects of species size were eliminated) and of species size (when the effects of food intake were removed). However, species with high (low) efficiencies have significantly higher (lower) reproductive rates. A lowering of the temperature from 25° to 15° C. increased the size of the species, increased food intake, but decreased reproductive rate. In all species except H. pseudoligactis a corresponding increase in the production of bud energy with no change in efficiency also occurred. On the other hand, lowering of the temperature for H. pseudoligactis significantly lowered reproductive efficiency but had no effect on the total calorific output of buds. This species, in constrast to the others, appears to have a compensatory ability to adjust its efficiency to maintain a high calorific output when temperature increases. It was also found that albino Chlorohydra have budding efficiencies of around 35 percent which are not influenced by changes in food intake or light. Normal green hydras, however, have efficiencies which range from 40 to 62 percent above their albino counterparts when fed once a day and once every two days in light respectively. It it concluded first, that the symbiotic algae in the gastrodermals cells of green hydra contribute quantitatively in the order of the above amounts to the growth process in this species, and second, that green hydras have the ablity to increase their growth efficiency when food intake is reduced thus reducing the drop in calorific but output that normally occurs in the albino (control) form.     

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.     

Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium “Chlorochromatium aggregatum”

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium “Chlorochromatium aggregatum”. The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and γ-carotene and OH-γ-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (≤94.6% sequence homology). The novel isolate is therefore described as a novel species within the genus Chlorobium, Chlorobium chlorochromatii.     

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。     

Bacterial symbionts on green hydra and their effect on phosphate uptake

Gram-negative rod-shaped bacteria (1.5–2m long and 0.5m wide) have been found associated with green hydra. They are always present on the hydra surface delineating the ectodermal cells, on animals in culture, and also on those sampled from a natural habitat. The bacteria could be removed by a 30-min treatment with antibiotics (50/ml polymyxin B and 50/ml streptomycin). Antibiotic-treated hydra took up 55% less phosphate from the medium than control hydra. The nutritional relationship between the bacteria and green hydra and possible routes of infection of the hydra by these prokaryotic symbionts are discussed. Their importance in interpreting results of certain types of physiological experiments using aquatic organisms is emphasized.     

Metatrancriptomic analysis from the Hepatopancreas of adult white leg shrimp (<Emphasis Type="Italic">Litopenaeus vannamei)</Emphasis>

The amoeba, Mayorella viridis contains several hundred symbiotic green algae in its cytoplasm. Transmission electron microscopy revealed strong resemblance between symbiotic algae from M. viridis the symbiotic Chlorella sp. in the perialgal vacuoles of Paramecium bursaria and other ciliates. Although it is thought that the M. viridis and symbiotic algae could be model organisms for studying endosymbiosis between protists and green algae, few cell biological observations of the endosymbiosis between M. viridis and their symbiotic algae have been published. In this study, we characterized the specificity of endosymbiotic relationships between green algae and their hosts. Initially, we established stable cultures of M. viridis in KCM medium by feeding with Chlorogonium capillatum. Microscopic analyses showed that chloroplasts of symbiotic algae in M. viridis occupy approximately half of the algal cells, whereas those in P. bursaria occupy entire algal cells. The symbiotic algae in P. bursaria contain several small spherical vacuoles. The labeling of actin filaments using Acti-stain? 488 Fluorescent Phalloidin revealed no relationship between host actin filaments and symbiotic algal localization, although the host mitochondria were localized around symbiotic algae. Symbiotic algae from M. viridis could infect algae-free P. bursaria but could not support P. bursaria growth without feeding, whereas the original symbiotic algae of P. bursaria supported its growth without feeding. These data indicated the specificity of endosymbiotic algae relationships in M. viridis and P. bursaria.     

中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

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.     

Lipid composition ofAcetabularia mediterranea (Lamour.) Variations during cap morphogenesis

Summary YoungAcetabularia mediterranea cells without cap have a fatty acid composition different from other green algae currently used for biological research. They contain important quantities of palmitic and oleic acid, but are very poor in polyunsaturated fatty acids such as linoleic and linolenic acid. (These polyunsaturated fatty acids are predominant in higher plants and many green algae.)     

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 endosymbiotic unit ofClimacostomum virens andChlorella sp. I. Morphological and physiological studies on the algal partner and its localization in the host cell

Summary The ciliateClimacostomum virens forms an endosymbiotic association with coccoid chlorophycean algae which can be isolated and grown as sterile mass cultures with an inorganic medium. According to both morphological and physiological properties, the algae probably belong to the genusChlorella and have some features in common with symbiotic chlorellae isolated from the ciliatesParamecium bursaria andStentor polymorphus. These and other endosymbiotic chlorellae studied so far excrete maltose or glucose, but algae fromClimacostomum virens show a different excretion pattern by releasing glucose, fructose and xylose. Possible biosynthetic pathways are discussed. Algae inClimacostomum virens are either located individually in special perialgal vacuoles where they are probably protected against attack by host lytic enzymes or to a lesser extent in food vacuoles in different states of digestion. The endosymbiotic character of theClimacostomum virens-Chlorella sp.-association is discussed.Dedicated to Prof. H. A.von Stosch on the occasion of his 75th birthday.     

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.     

Comparison of two aposymbiotic ciliate clones of Climacostomum virens by their ability for reinfection

The ability of two aposymbiotic (algae-free) subclones of the same green clone of C. virens to establish a stable symbiotic association with Chlorella sp. has been studied by light and electron microscopy. Alga-free subclone No. 1 was obtained from the original green clone by a long-term cultivation in darkness, while subclone No. 2 originated from one cell that spontaneously lost the algae and was found among normal green cells during daily inspection. For infection, algae isolated from ciliates with chlorellae of parental clone of C. virens were used. 5-10 minutes after feeding with Chlorella, specimens of both subclones show numerous algae mostly inside food vacuoles, but some rare algae (3-4 per cell) may occur in individual perialgal vacuoles. Later on, the number of symbiotic chlorellae in ciliates of subclone No. 1 increased, and a stable symbiotic association was reestablished. Unlike, in specimens of subclone No. 2 all newly ingested algae were seen digested within food vacuoles. Within 24-28 h all the ciliates investigated appeared free of algae. However, obviously stable symbiotic ciliate-algae systems in this subclone were obtained after improving the microinjection technique. Injection of algae into alga-free ciliates resulted in maintenance of intact chlorellae in these ciliates. The algae were seen to be located individually within perialgal vacuoles, being presumably protected against host lytic enzyme attack. The endosymbiont population in ciliates was established from as many as 3-5 originally injected algae. The number of symbiotic chlorellae increased steadily reaching the value equal to that in the parental clone 28-30 days after the start of experiment.     

Secondary Carotenoids of Eremosphaera viridis De Bary (Chlorophyceae) Under Nitrogen Deficiency*

Under nitrogen deficiency the unicellular chlorococcalean green alga, Eremosphaera viridis De Bary, was able to synthesize secondary carotenoids (SC). Nine SC were identified as six astaxanthin esters, echinenone, canthaxanthin and a lutein ester, previously not described in green algae under nitrogen deficiency. These SC, jS-carotene and the main part of lutein were located in lipid bodies outside the chloroplasts in the cytosol. The synthesis of SC could be inhibited by the herbicides norflurazon and nicotine. This result supported the idea that SC in cells ot Eremosphaera viridis were synthesized de novo rather than derived from primary carotenoids.     

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.     

Noninvasive methodology for analyzing the response of the system to electromagnetic fields of industrial frequency

Characteristics of the main biochemical components in surface structures and whole cells of unicellular green algae D. tertiolecta and T. viridis before and after the influence of an electromagnetic field (EMF) of industrial frequency were obtained by the method of noninvasive analysis in nonpolarized light. We showed fundamental differences in response to the influence of EMF among cells of algae cultures, which manifest themselves in changes in the main biochemical components and their concentration gradients. These changes led to the transformation of metabolism.