Exploitation or cooperation? Evolution of a host (ciliate)-benefiting alga in a long-term experimental microcosm culture

Controversy persists as to whether the acquisition of beneficial metabolic functions via endosymbiosis can occur suddenly on an evolutionary time scale. In this study, an early stage of endosymbiotic associations, which evolved from previously unassociated auto (photo)- and heterotrophic unicellular organisms was analyzed using an experimental ecosystem model, called CET microcosm. This ecosystem model was composed of a green alga (Micractinium sp.; formerly described as Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). Our previous study using a CET microcosm that was cultured 3–5 years revealed that fitness of the ciliate increased by harboring algal cells within its own cells. This fact suggested three possibilities: (i) the ciliate evolved the ability to exploit intracellular algal cells (“exploiter ciliate hypothesis”), (ii) the alga evolved the ability to benefit the host ciliate by providing photosynthates (“cooperator alga hypothesis”), and (iii) a combination of (i) and (ii). To test these hypotheses, two-by-two co-cultures were conducted between the ancestral or derived ciliate and the ancestral or derived alga. The experimental results demonstrated that a cooperative alga evolved in the microcosm, although the possibility remains that an exploitative genotype of the ciliate might also exist in the population as a polymorphism. Remarkably, an algal isolate prolonged the longevity of not only the isolated ciliate, but also the ancestral ciliate. This result suggests that once a cooperative algal genotype evolves in a local population, it can then be transmitted to other individuals of the prospective host species and spread rapidly beyond the local range due to its positive effect on the host fitness. Such transmission suggests the possibility of a sudden acquisition of beneficial autotrophic function by the pre-associated host.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Astaxanthin production by a green alga,Haematococcus pluvialis accompanied with morphological changes in acetate media

Two acetate containing media were developed for astaxanthin production by a green unicellular alga, Haematococcus pluvialis. The basal medium, a vegetative growth medium facilitated the algal cell growth, whereas the modified medium was likely to induce morphological changes with the formation of large cysts and bleached cells which seemed to consequently enhance the carotenoid biosynthesis. In the two-stage culture, the injection of ferrous ion with acetate into the basal medium on the fourth day, was greatly stimulative for both the algal cell growth and the astaxanthin formation at a high light intensity. In addition, carotenoid precursors, mevalonate and pyruvate were effective on the carotenoid formation in the modified medium. Pyruvate was an especially good carbon source both for the algal cell growth and the carotenoid synthesis.     

Self-supporting artificial system of the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and the ascomycetous fungus <Emphasis Type="Italic">Alternaria infectoria</Emphasis>

A long-living (of up to several years) bipartite system was constructed between the unicellular green alga Chlamydomonas reinhardtii and the ascomycetous fungus Alternaria infectoria. The metabolic cooperation between the two organisms was tested with infecting A. infectoria hyphae into nitrogen starving yellow C. reinhardtii culture. After the infection, a slow greening process of the algal cells was observed, which was studied by measuring the increasing chlorophyll content, the appearance of chlorophyll-protein complexes – using 77 K fluorescence spectroscopy, and the measurement of photosynthetic oxygen production. Transmission electron microscopy and laser scanning microscopy images showed that no direct physical contacts were formed between the algal cells and the hyphae in the long-living symbiosis but they were joint in a mucilaginous bed allowing diffusion processes for metabolic cooperation. The increased free amino acid content of the medium of the long-living bipartite cultures’ indicated possible nitrogen supply of hyphal origin, which allowed the re-greening of the algal cells. The results of this work underline the importance of symbiosis-like stable metabolic coexistence, which ensures survival under extreme environmental conditions.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Lysis of a red-tide causing alga,Alexandrium tamarense,caused by bacteria from its phycosphere

There are bacteria coexisting in xenic cultures of Alexandrium tamarense, a red-tide causing alga. However little is known concerning the interactions between the alga and the bacteria in its phycosphere. The objective of the current study was to determine the effect of the bacteria in its phycosphere on the growth of the alga. We added one percent (v/v) Zobell 2216E medium to A. tamarense culture to alter bacterial growth and the results showed that algal cells were all lysed within 14 h. After adding the medium, both the abundance and the extracellular enzyme activity of the bacteria increased by 50–100 times from the 4th to the 10th hour which resulted in lysis of the algae. The 16S rRNA gene fragments of the bacteria were amplified from the DNA extracted from A. tamarense cultures and analyzed by denaturing gradient gel electrophoresis and sequencing. The structure of the bacterial community in phycosphere changed significantly during algal lysis. Two bacterial genera, Alteromonas sp. and Thalassobius aestuarii sp. are key factors that caused the lysis, and the β-glucosidase and chitinase produced by the bacteria in the phycosphere could directly cause the lysis. These experiments provide evidence that bacteria in its phycosphere play a key role in the culture of A. tamarense, and may provide insights into the future biocontrol of red-tides.     

Infection of the heart cockle, Clinocardium nuttallii, from Yaquina Bay,Oregon, with an endosymbiotic alga

Clinocardium nuttallii from Yaquina Bay, Oregon, were found to harbor an endosymbiotic alga. Some aspects of this relationship are presented. The areas of infection include the siphon, mantle, and occasionally the foot. C. nuttallii under 2 years old are not infected; the incidence and intensity of the infection increases in the older age groups. For all ages the incidence averages 35%. Pigment composition, morphology, and growth characteristics of the alga are similar to those found in the genus Chlorella. In situ, the algae form dense, sometimes massive colonies but do not appear to cause any host reaction or enfeeblement. The thick layers surrounding the algal cells in situ, the dense colonies, and the in vitro reaction to host extract suggest the alga is parasitic. However, the presence of chloroplasts, the location of the algal cells only in illuminated tissue, the seemingly unhampered reproduction in situ, and the eventual adaptation to mineral medium suggest the alga is a facultative parasite. Experimental infection was achieved by feeding mature uninfected cockles a diet of only symbiont cells. In vitro observations found the symbiont cells readily engulfed by host blood amoebocytes. It is believed that the animal acquires the infection through phagocytosis of the symbiont cells and subsequent diapedesis across epithelial barriers by host amoebocytic cells.     

ENTRY OF A CHLORELLA-VIRUS INTO ITS HOST CELL1

The endosymbiotic Chlorella sp. (Chlorophyceae) of Paramecium bursaria (Ciliata) can be infected by a double-stranded DNA-containing virus (Chlorella-virus) that has a phagelike entry mechanism. Electron micrographs show that soon after attachment of the virus to the algal cell wall, a hole is formed through which the viral DNA enters the alga. Biochemical studies on a European Chlorella-virus system suggest that digestion of the algal cell wall is caused by glycolytic enzymes, one of which was identified as a β-d -glucosidase. Enzymes are bound to the virus capsid and are activated only after or by the attachment of the virus to its cognate alga or to preparations of the algal cell wall. Common features of viral cell wall-digesting enzymes and algal autolysins are discussed.     

Modelling microalgal activity as a function of inorganic carbon concentration: accounting for the impact of pH on the bicarbonate system

The aim of this study is to develop a generic model that can predict algal photosynthetic activity as a function of total inorganic carbon and pH, which will assist in the design and operation of algal culture systems. This is important as the availability of inorganic carbon plays a critical role in algal growth and product accumulation, and in practice, pH is not constant in an algal culture. Hence, such a model will assist in predicting and understanding carbon limitation in algae growth and product accumulation systems. The model builds on published work on inorganic carbon uptake in natural algal systems and extends it to systematically account for wide pH and total inorganic carbon ranges. This study develops and validates a simple model which integrates a summative carbon dioxide and bicarbonate Monod kinetic relationship with inorganic carbon equilibrium chemistry. This is the first time that the algal photosynthetic oxygen production rate has been described as a function of both total inorganic carbon and pH. The model was tested against published and experimental data over an extended pH and total inorganic carbon range. Kinetic parameters estimated by the model match those presented in the literature. The Chlorella alga tested in the experiments showed little affinity for bicarbonate which agrees with previous observations for this alga.     

The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

Interactions between the unicellular red alga Rhodella reticulata (Rhodophyta) and contaminated bacteria

AIMS: To define the role of the bacterial strains LR1 and LR3 in the Rhodella cell destruction caused by Cytophaga sp.LR2. METHODS AND RESULTS: The bacteria were obtained from algal culture with destruction. They were isolated in pure culture and tested for biochemical activities using Polymicrotest. The ability of bacteria to degrade and utilize the algal polysaccharide was investigated. The bacteria were grown in a media containing Rhodella polysaccharide as a sole carbon source. The level of the reducing sugars in the culture media was determined. Scanning electron microscopy (SEM) was used to define the location of bacteria in extensively and intensively cultivated Rhodella reticulata previously infected by Cytophaga sp. LR2. CONCLUSIONS: The lysis of Rhodella reticulata cells is due to the joint action of the three bacterial strains with the former pathogen Cytophaga sp. LR2 playing the main role. The accumulation of the polysaccharide and the excreted metabolites of the strains LR1 and LR3 stimulated the development of Cytophaga sp. LR2. The adaptation of the strain to particular conditions of alga cultivation and the utilization of polysaccharide as a sole carbon source supported its stable growth in alga suspension and destruction of Rhodella cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The predominance of Cytophaga sp. LR2 over the two other contaminants and the lysis of Rhodella reticulata cells resulted from the ability of the bacterium to attach to the algal polysaccharide sheath. The formation of slime and extrusions facilitated the phenomenon of bacterial adhesion to the algal surface as well as the formation of colonial alga - bacterial spherules. The sedimentation of these aggregates decreased the ability of the algal strain to photosynthesize, led to the lysis of the cells and finally caused the death of Rhodella.     

Growth and production of cell constituents in batch cultures of botryococcus sudeticus

The growth of, and the production of neutral lipids, carbohydrates and proteins by, the alga Botryococcus sudeticus in batch culture is described. The algal mass contains, during the stationary phase of growth, about 4.5% protein, 7.5% carbohydrate and 22.0% neutral lipid on a dry weight basis. Some physiological characteristics of this species are discussed.     

Evidence for the presence of non-receptor protein tyrosine kinases in algal cells

Two orders of green alga (Cladophorales and Charales) were investigated for the presence of protein tyrosine kinase activity. Proteins of 70 and 85 kDa were found to be tyrosine phosphorylated in Cladophora fracta, with an additional phosphorylated band evident at the 120-kDa region in Chara vulgaris, suggestive of the presence of putative tyrosine kinase activity in these algal species. A 70-kDa protein was immunoprecipitated from both species using a polyclonal antibody against non-receptor protein tyrosine kinase Syk. The protein was found to be phosphorylated on tyrosine, which was prevented upon pretreatment of algal cells with piceatannol. The extent of phosphorylation directly correlated with algal growth, suggesting a link between Syk kinase activity and growth signaling. These observations supported the presence of Syk-like kinase in the green algal species, which could have critical role in the algal growth and development.     

An improved cheap culture medium for the blue-green microalga Spirulina

Summary The possibility of using crude seasalt, and saltpeter as the basis of a culture medium for the blue-green alga Spirulina maxima, was investigated. Saltpeter appears to be an excellent nitrogen and micronutrient-source and is very cheap. The effect of the nutrient level on algal growth, and nitrogen conversion efficiency, was studied. The results suggest the possibility of using this medium in a large-scale algal culture.     

Virus-Specific Responses of Heterosigma akashiwo to Infection

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.     

Studies on inoculation of aerial algae. I. Semi-mass culture and quantification of algal color of Klebsormidium flaccidum (Ulotrichales, Chlorophyceae) sprayed on to concrete board

This paper describes methods of semi-mass culture and the quantification of algal color sprayed on a concrete board of the alga Klebsormidium flaccidum (Küitzing) S. Mattox et Blackwell. The alga was cultured in 100 L tanks normally used for hatching brine shrimp, with aeration from the deepest parts of the slanting sides. Initially mean algal density increased rapidly and attained 9.1 times the original density after 4 weeks. Subsequently, increase of the algal density was slow, and the density became 16.1 times the original level after 8 weeks. The alga cultured in this study was sprayed on to a concrete board using an agricultural sprayer. The relationship between the algal density on the board and its color, expressed as L*a*b* color space, was quantified. The chromaticity was maximum as the algal density on the board was about 5 g/m², and the color was most vivid. When the algal density was more than 20 g/m², both the lightness and chromaticity changed slightly, and the color became dark green.     

Effects of acidic conditions on the physiology of a green alga (Micractinium sp.) before and after a 5-year interaction with Tetrahymena thermophila in an experimental microcosm

The mechanisms through which algae evolve physiological characteristics related to endosymbiotic associations with heterotrophic organisms remain unclear. We previously showed that a green alga (Micractinium sp.) was able to evolve a host (ciliate)-benefiting phenotype that prolonged the longevity of Tetrahymena thermophila in the absence of bacteria during a 5-year culture period in an experimental microcosm. Comparative experiments between the ancestral alga (i.e. original) and evolved algal clones of the same lineage can be performed to analyse the mechanisms that underlie algal evolution during interactions with ciliates. Here, we investigated the effects of acidic conditions on algal physiology because the acidic conditions within the food vacuoles of Tetrahymena could potentially affect algal evolution during long-term interactions. It might be expected that algal clones isolated from T. thermophila hosts would have developed the ability to resist acidic conditions in order to establish within the host, when compared with the ancestral strain. Unexpectedly, comparative analyses demonstrated that acidic conditions with pH values ranging from 3.9 to 4.3 limited the growth of the isolated algal clones SC9-1 and SC10-2, whereas the ancestral strain could grow under these conditions. Acidic conditions were responsible for significantly lower chlorophyll a/chlorophyll b ratios in the isolated algal clones, and the isolated clone SC9-1 exhibited a high cellular content of chlorophyllide a during a short exposure to acidic conditions, suggesting that degradation of chlorophyll a occurred. Furthermore, acidic conditions increased the release of extracellular glycerol and sucrose in the SC9-1 clone. These results indicated that the ancestral strain showed phenotypic changes related to acidic conditions, which may reflect the ongoing development of an algal phenotype suited to symbiosis. Hypotheses are proposed to explain the limited growth of the isolated clones, and implications for the Micractinium–Tetrahymena association are discussed.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Metal metabolism in the red alga Cyanidium caldarium and its relationship to metal tolerance

The unicellular red alga Cyanidium caldarium is tolerant to high levels of various metal ions. Cells of this alga cultured with divalent metal ions at 5 mM contained an elevated concentration of each metal, with the highest level for Zn followed by Mn > Ni > Cu. This order is in fair agreement with the toxicity levels reported previously, with the exception of Mn, which shows a toxicity level comparable to that of Ni. Transmission electron microscopy indicated the presence of electron-dense bodies in the algal cells, and elemental analysis by energy dispersive X-ray spectrometry showed high levels of Fe and P in these bodies. Accumulation of Zn was found in these particles in Zn-treated algal cells, whereas no such deposition was found for Cu, Ni, or Mn in cells treated with the respective metals. Although trapping of Zn in the intracellular bodies may contribute to reduction of metal activity in the cells, this effect can be overcome by high intracellular levels of Zn that result in a high degree of toxicity. The correlation between intracellular concentration and toxic levels of metal ions implies that the reduced incorporation of the metals is a major detoxification mechanism in this alga.