Juyun Lee

Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy

Katano T., Yoshida M., Lee J., Han M.-S. and Hayami Y. 2009. Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy. Phycologia 48: 473–479. DOI: 10.2216/08-102.1. Chattonella antiqua and C. marina are harmful algal species that cause massive fish kills in coastal environments. Generally, Chattonella cells cannot be preserved well by fixation because of their fragile nature. In the present study, we developed a new fixative for Chattonella. Phosphate-buffered saline, which is generally used as a buffer for fixative, caused precipitation after fixation. In contrast, Hepes or sodium cacodylate prevented the precipitation. Moreover, these buffers worked well in the fixation to preserve cell morphology. Since Hepes is not as noxious as cacodylate, we selected Hepes as the buffer for the fixative. Cell counting revealed that the decrease in cell numbers by fixation was negligible and did not start until at least 8 days after fixation. We successfully analysed the DNA amount in fixed cells of Chattonella by flow cytometry. The present study demonstrated that Hepes-buffered paraformaldehyde and glutaraldehyde are superior to other fixatives for flow cytometry and light microscopy.

Comprehensive comparisons of three pennate diatoms, Diatoma tenuae, Fragilaria vaucheriae, and Navicula pelliculosa, isolated from summer Arctic reservoirs (Svalbard 79°N), by fine-scale morphology and nuclear 18S ribosomal DNA

Here we report morphological and molecular characteristics of dominant freshwater diatoms in summer Arctic reservoirs of Svalbard (Norway), using four culture isolates, when we collected the samples in the field on 15 August 2005. Analyses of morphology and BLAST searches with 18S rDNA sequences identified them to Diatoma tenue (HYNP006, HYNP013), Navicula pelliculosa (HYNP021), and Fragilaria vaucheriae (HYNP022), respectively. Comparative studies of morphology revealed that the body shapes of the three polar diatoms were nearly identical to the known morphology of each species; however, they were considerably shorter in body length than previously described identical species from other locations. The 18S rDNA sequences of the diatoms were nearly identical to the same species from temperate and other regions. Phylogenetic analysis showed that the polar diatoms each formed a clade with their identical species and genera according to their taxonomic positions. This suggests that the polar diatoms may possess little or no genetic or morphological variation compared to more temperate strains.

Effects of Temperature and Salinity on the Growth and the Optimum Nitrogen to Phosphorus Ratio for the Culture of Diatoma tenue Isolated from a Temporary Arctic Pond in Svalbard, Norway

ABSTRACT We investigated the effects of temperature and salinity on the growth of Diatoma tenue isolated from a temporary arctic pond in a high-latitude region of Svalbard, Norway. We also examined the optimum nitrogen to phosphorus (N:P) ratio of the culture media. The highest growth rate (0.38 day−1) of D. tenue was achieved at 15°C, while growth was completely inhibited above 20°C. D. tenue could not grow when cultured at above 5%o salinity. The final cell density of D. tenue was increased with an increasing phosphate concentration in the culture media from 0 to 5 μmol P L−1. The final cell density became stable at above 5 μmol P L−1. The N:P molar ratio of the media containing 5 μmol P L−1 was 34.8. These results show that D. tenue growth is limited by phosphorus at N:P ratios greater than 34.8, with a nitrogen limitation below 34.8. This finding indicates that this alga has a higher optimum N:P ratio than the Redfield ratio of 16.

Monitoring the seasonal dynamics of microalgae in the South Sea of Korea by use of a cytochrome c oxidase I DNA barcode

Microalgae are important primary producers in the marine environment, and are also important sources of health foods and medical products. Rapidly growing microalgae have potential use for the production of commercial products, but can also cause harmful microalgal blooms in natural ecosystems. There are many available techniques for the identification of microalgae in natural ecosystems. We used the mitochondrial cytochrome c oxidase subunit I gene as DNA barcode to identify 14 species of microalgae from the South Sea of Korea and to discriminate among similar biogeographic subgroups within species. In addition, we designed species-specific primers targeting the cytochrome c oxidase subunit I gene to evaluate monthly changes of microalgae throughout the year. Chaetoceros brevis, Asterionellopsis glacialis and Stephanopyxis turris were present during all seasons, whereas Skeletonema japonicum, Nitzschia improvisa, Ditylum brightwellii and Chaetoceros diadema were only detected during winter and spring. Our results indicate that species-specific polymerase chain reaction of the cytochrome c oxidase subunit I gene can be used to monitor the seasonal dynamics of microalgae in the South Sea of Korea. This polymerase chain reaction detection method successfully identified the 14 most common species of microalgae with the same polymerase chain reaction condition near Tongyeong, in the South Sea of Korea.

Effect of Temperature on Inorganic Carbon Acquisition of Chlamydomonas reinhardtii

ABSTRACT Carbon dioxide availability for microalgae in aquatic environments increases with decreasing water temperature, while photosynthetic activity generally decreases. Therefore, inorganic carbon acquisition by algal cells is greatly affected by temperature. We investigated half-saturation constants [Km(DIC), Km(CO2)] of inorganic carbon in photosynthesis under various temperatures for a strain of Chlamydomonas reinhadtii. C. reinhardtii showed an active carbon concentrating mechanism (CCM) at all temperature conditions investigated (5–25°C), implying that CCM activity is not diminished at low temperatures. The maximum photosynthetic rate was recorded at 15°C, while maximum CCM activity was detected at 20°C. A higher optimum temperature for CCM activity than for photosynthesis may compensate for lower photosynthetic rates above the optimum temperature. CCM may play a more significant role at higher temperatures in algal photosynthesis in aquatic environments.

Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra

Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization (NPA-SH) probe that targets the large subunit of ribosomal RNA (LSU rRNA). We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H. triquetra at a concentration of 1.5×104 cells/mL, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015 (3.0×104 cells/mL). We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H. triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.

Evaluation of rapid cell division in non-uniform cell cycles.

To better understand the mechanisms of development of harmful algal blooms (HABs), accurate estimates of species‐specific in situ growth rates are needed. HABs are caused by rapid cell division by the causative microorganisms. To accurately estimate the in situ growth rates of harmful algae having non‐uniform and/or irregular cell cycles, we modified a standard equation based on the cell cycle, and calculated the in situ growth rate to describe the process of bloom development in nature. Sampling of a developing bloom of Heterosigma akashiwo in Pohang Bay, Korea, was conducted every 3 h from 15:00 on August 2 to 07:00 on August 4, 2006. The amount of H. akashiwo DNA was measured using flow cytometry following tyramide signal amplification‐fluorescence in situ hybridization. On August 2, the percentage of G1 phase cells decreased from 15:00 to 19:00 then increased until 22:00; it then decreased until 07:00 on August 3, followed by an increase to 10:00. This indicates the ability of the cells in nature to undergo more than one round of division per day. During the following night two rounds of division did not occur. The in situ growth rates estimated using the modified equation ranged from 0.31 to 0.53 d−1. We conclude that the use of this equation enables more accurate estimates of bloom formation by rapidly dividing cells.

Expression of Mycosporine-like Amino Acids Biosynthetic Genes in the Chlamydomonas sp. Exposed to Radiofrequency

Mycosporine-like amino acids (MAAs) are UV-absorbing substances, and diverse marine organisms have the evolved the capacity to diminished the direct and indirect damaging effects of environmental ultraviolet radiation by synthesis and accumulation of MAAs. In this study, we manufactured a radiofrequency (RF) generation device and applied to microalgal culture. 0.35±0.05 mHz of RF was supplied to culture vessel for Chlamydomonas sp. and samples were harvested at the designated time intervals (1, 0.5, 1 and 2 hr). MAAs biosynthetic genes, dehydroquinate synthase homolog (DHQS-like) and nonribosomal peptide synthetase homolog (NRPS-like), were cloned from Chlamydomonas sp. and their gene expressions under the RF exposure were analyzed using qRT-PCR. DHQS-like and NRPS-like gene expressions of Chlamydomonas sp. exposed to RF were increased 1.46 and 1.19 fold at 1 hr, respectively. These results means that DHQS-like and NRPS-like genes can be good biomarker candidates for diagnosis of MAAs biosynthesis in the Chlamydomonas sp.

Expressed Sequence Tag Analysis of Toxic Alexandrium tamarense and Identification of Saxitoxin Biosynthetic Genes

Expressed sequence tag (EST) library was constructed from A. tamarense. Base sequences of EST clones were analyzed and saxitoxin biosynthesis-related genes were cloned. Sequences of 827 clones were analyzed and 564 EST were functionally clustered using Blast searches against GenBank. Main genes in the EST had functions on cellular organization, cell metabolism, energy, cell cycle and DNA processing, cellular transport and transport, cell rescue, defense, death and aging, and transcription. Moreover, expression of S-adenosylmethionine synthetase and H2A histone family genes were increased in the toxic A. tamarense. These results show that two genes could be a good biomarkers for the detection of saxitoxin biosynthesis in the A. tamarense.