Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.

ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m-2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO43- uptake was more serious compared to NO3- uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca2+-ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca2+-ATPase suppression, and a relation between Ca2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the glutathione cycle was involved in the response of marine microalgae to UV-B stimuli.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

Morphology and phylogeny of a new wall-less freshwater volvocalean flagellate, Hapalochloris nozakii gen. et sp. nov. (Volvocales, Chlorophyceae).

New strains of a wall-less unicellular volvocalean flagellate were isolated from a freshwater environment in Japan. Observations of the alga, described here as Hapalochloris nozakii Nakada, gen. et sp. nov., were made using light, fluorescence, and electron microscopy. Each vegetative cell had two flagella, four contractile vacuoles, and a spirally furrowed cup-shaped chloroplast with an axial pyrenoid, and mitochondria located in the furrows. Based on the morphology, H. nozakii was distinguished from other known wall-less volvocalean flagellates. Under electron microscopy, fibrous material, instead of a cell wall and dense cortical microtubules, was observed outside and inside the cell membrane, respectively. Based on the phylogenetic analyses of 18S rRNA gene sequences, H. nozakii was found to be closely related to Asterococcus, Oogamochlamys, Rhysamphichloris, and "Dunaliella" lateralis and was separated from other known wall-less flagellate volvocaleans, indicating independent secondary loss of the cell wall in H. nozakii. In the combined 18S rRNA and chloroplast gene tree, H. nozakii was sister to Lobochlamys.

Controlled regular locomotion of algae cell microrobots.

Algae cells can be considered as microrobots from the perspective of engineering. These organisms not only have a strong reproductive ability but can also sense the environment, harvest energy from the surroundings, and swim very efficiently, accommodating all these functions in a body of size on the order of dozens of micrometers. An interesting topic with respect to random swimming motions of algae cells in a liquid is how to precisely control them as microrobots such that they swim according to manually set routes. This study developed an ingenious method to steer swimming cells based on the phototaxis. The method used a varying light signal to direct the motion of the cells. The swimming trajectory, speed, and force of algae cells were analyzed in detail. Then the algae cell could be controlled to swim back and forth, and traverse a crossroad as a microrobot obeying specific traffic rules. Furthermore, their motions along arbitrarily set trajectories such as zigzag, and triangle were realized successfully under optical control. Robotize algae cells can be used to precisely transport and deliver cargo such as drug particles in microfluidic chip for biomedical treatment and pharmacodynamic analysis. The study findings are expected to bring significant breakthrough in biological drives and new biomedical applications.

A Microreactor System for Cultivation of Haematococcus pluvialis and Astaxanthin Production.

Development of efficient culture and monitoring system for cell growth and production of useful materials is required for practical utilization of microalgae. In the present study, we developed a PDMS-based microreactor system for efficient, rapid culture of microalgae and monitoring of cell growth, carotenoid content under diverse culture conditions. Due to advantages of PDMS, we optimized culture conditions (light intensity, pH, nitrate depletion, carbon dioxide concentration) for improving growth rate and astaxanthin productivity in considerably less time compared to conventional culture methods using flask or well plate. In addition, we found that there was a strong linear correlation between fluorescence intensity of astaxanthin stained by Nile red and the astaxanthin content, which can be utilized as a high-throughput screening tool in microfluidic systems. In this study, the growth rate of vegetative Haematococcus pluvialis was improved by 60% in microfluidic chamber than in flask and astaxanthin was produced up to 362 mg/L under the optimal conditions (300 µmol photon/m2/s of light, 7% CO2 (v/v), and pH 7.0) using designed microfluidic devices. This result shows that microfluidic system can provide effective means to address development of microalgal strains including H. pluvialis and bioprocess.

Characterization and expression patterns of nitrate reductase from Dunaliella bardawil under osmotic stress and dilution shock.

A complementary DNA (cDNA) of nitrate reductase (NR) from Dunaliella bardawil was isolated using RT-PCR and RACEs techniques. The full-length D. bardawil NR (DbNR) cDNA is 3,107 bp containing a putative open reading frame of 2,670 bp in length which encodes 889 amino acids with a calculated molecular weight (MW) of 98.37 kDa, a 34-bp 5'-untranslated region, and a 3'-untranslated region of 403 bp with a poly (A) tail. BLAST search showed that the nucleotide and putative protein sequence exhibit sequence identities of 92 and 79% with the corresponding gene from Dunaliella tertiolecta, respectively. Protein structural analysis showed a typical NR structure of DbNR with five structural distinctive domains which form three common subparts of eukaryotic NR (Euk-NR). Phylogenetic analysis based on the holo-DbNR and sulfite oxidase (SO) and cytochrome b reductase (CbR) subparts manifested that (1) DbNR has a closer relationship with those counterparts from algae and higher plants than from other species and (2) DbNR might have evolved from ancient SO and CbR in a "domain shuffling" pattern. The glycerol contents and transcriptional expression patterns of DbNR under salt stress and dilution shock treatments were also traced. The results implied an indirect role of NaCl on the induction of DbNR through an osmoregulation pathway.

Organelle genome complexity scales positively with organism size in volvocine green algae.

It has been argued that for certain lineages, noncoding DNA expansion is a consequence of the increased random genetic drift associated with long-term escalations in organism size. But a lack of data has prevented the investigation of this hypothesis in most plastid-bearing protists. Here, using newly sequenced mitochondrial and plastid genomes, we explore the relationship between organelle DNA noncoding content and organism size within volvocine green algae. By looking at unicellular, colonial, and differentiated multicellular algae, we show that organelle DNA complexity scales positively with species size and cell number across the volvocine lineage. Moreover, silent-site genetic diversity data suggest that the volvocine species with the largest cell numbers and most bloated organelle genomes have the smallest effective population sizes. Together, these findings support the view that nonadaptive processes, like random genetic drift, promote the expansion of noncoding regions in organelle genomes.

Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga.

Nitrogen availability and light intensity affect ß-carotene overproduction in the green alga Dunaliella salina. Following a previous study on high-light stress, we here report on the effect of nitrogen depletion on the growth characteristics and ß-carotene as well as fatty acid metabolism of D. salina under a constant light regime in a turbidostat. Upon nitrogen depletion, the biomass yield on absorbed light approximately doubled, due to a transient increase in cell division rate, swelling of the cells and a linear increase of the density of the cells. Simultaneously, ß-carotene started to accumulate up to a final intracellular concentration of 14 mg LCV⁻¹ (i.e. 2.7% of AFDW). This ß-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates ß-carotene in lipid globules, we also determined the fatty acid content and composition of D. salina. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation, indicating that ß-carotene and total fatty acid accumulation were unrelated, similar to what was found previously for high-light treated cells. However, for both high-light and nitrogen stress, ß-carotene accumulation negatively correlated with the degree of unsaturation of the total fatty acid pool and, within the individual fatty acids, correlated positively with oleic acid biosynthesis, suggesting that oleic acid may be a key component of the lipid-globule-localized triacylglycerols and thereby in ß-carotene accumulation.

Production, extraction, and quantification of astaxanthin by Xanthophyllomyces dendrorhous or Haematococcus pluvialis: standardized techniques.

For many years, benefits and disadvantages of pigments production either by microalgae or yeasts have been under analysis. In this contribution we shall deal with Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and Haematococcus pluvialis, which are known as major prominent microorganisms able to synthesize astaxanthin pigment. Then, the usual trend is to look for optimal conditions to conduct astaxanthin synthesis. From one side, pigment production by H. pluvialis is promoted under cellular stress conditions like nutrient deprivation, exposition to high light intensity, aeration. On the other side, X. dendrorhous is able to show significant increase in astaxanthin synthesis when grown in natural carbon sources like coconut milk, grape juice. The main aim of this chapter is to describe optimal environmental conditions for astaxanthin production by X. dendrorhous or H. pluvialis.

Molecular cloning and characterization of a vacuolar H+₋pyrophosphatase from Dunaliella viridis.

The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na(+). In plants, sequestration of Na(+) into the vacuole is driven by the electrochemical H(+) gradient generated by H(+) pumps, and this Na(+) sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H(+) pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V-H(+)-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V-H(+)-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V-H(+)-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na(+) hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V-H(+)-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V-H(+)-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V-H(+)-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.

Application of terahertz time-domain spectroscopy in intracellular metabolite detection.

Using terahertz time-domain spectroscopy (THz-TDS), we have investigated the THz spectra of astaxanthin and riboflavin and the spectra of two kinds of cell, haenatcoccus plusivalis and bacillus subtilis, which could produce astaxanthin and riboflavin, respectively, during their metabolite process. Riboflavin was found to be much more absorptive to THz radiation and have richer spectral characteristics than astaxanthin. As an intracellular metabolite, riboflavin could be distinguished from the cells by using THz-TDS. The technique has potential applications in high-throughput screening of industrial strains.

Germ cell specification in Volvox carteri.

Volvox carteri illustrates with diagrammatic clarity Weismann's concept of an immortal germline that produces a mortal soma that will carry it for a time, but then perish. Each V. carteri adult consists of about 16 asexual reproductive cells (gonidia) in the interior of a sphere that consists at its surface of about 2000 biflagellate somatic cells. When mature, each gonidium divides to form a juvenile with this same cellular composition. Half-way through their maturation, juveniles hatch out of the parenteral spheroid, whereupon parental somatic cells undergo programmed death while juvenile gonidia prepare for a new round of reproduction. The first visible step in V. carteri germ-soma differentiation is asymmetric cleavage, which sets apart large gonidial initials from small somatic initials. Experimental analysis indicates that it is a difference in size, not any difference in cytoplasmic quality, that determines whether a cell will become germinal or somatic. Mutational and molecular studies lead to the following model for the genetic control of the germ-soma dichotomy: first, the gls locus acts to cause asymmetric division; then large cells activate a set of lag loci that suppress expression of somatic genes, while small cells activate the regA locus that suppresses gonidial genes.

The relationship between cell size and cell fate in Volvox carteri.

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.