Mikihide Demura

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Metagenomics approaches have been developing rapidly in marine sciences. However, the application of these approaches to marine eukaryotes, and in particular to the smallest ones, is challenging because marine microbial communities are dominated by prokaryotes. One way to circumvent this problem is to separate eukaryotic cells using techniques such as single-cell pipetting or flow cytometry sorting. However, the number of cells that can be recovered by such techniques remains low and genetic material needs to be amplified before metagenomic sequencing can be undertaken. In this methodological study, we tested the application of whole-genome amplification (WGA) to photosynthetic eukaryotes. We performed various optimization steps both on a mixture of known microalgal strains and on natural photosynthetic eukaryote populations sorted by flow cytometry. rRNA genes were used as markers for assessing the efficiency of different protocols. Our data indicate that WGA is suitable for the amplification of photosynthetic eukaryote genomes, but that biases are induced, reducing the diversity of the initial population. Nonetheless, this approach appears to be suitable for obtaining metagenomics data on microbial eukaryotic communities.

Taxonomic revision of Chattonella antiqua, C. marina and C. ovata (Raphidophyceae) based on their morphological characteristics and genetic diversity

Demura M., Noël M.-H., Kasai F., Watanabe M.M. and Kawachi M. 2009. Taxonomic revision of Chattonella antiqua, C. marina and C. ovata (Raphidophyceae) based on their morphological characteristics and genetic diversity. Phycologia 48: 518–535. DOI: 10.2216/08-98.1. Three raphidophyte species, Chattonella antiqua, C. marina and C. ovata, are red tide–forming harmful phytoplankton species, causing mass mortality at fish farms. These species were originally distinguished from each other solely based on their morphological characters. Recent genetic diversity analyses showed that the three species were extremely similar. It was therefore necessary to re-examine these three species to determine if they were independent single species. We compared 104 strains of the three species using morphological characters, nuclear ITS rDNA regions, the chloroplast rbcL gene, the mitochondrion COI gene and selected microsatellite regions. The morphological characters for the three species formed a continuum of variation instead of clearly defined limits. Compared with other selected heterokontophytes, their genetic divergence in the analyzed three regions was found to be at the intraspecies level. Microsatellite markers distinguished each of the 104 strains, but the strains apparently formed a single geographical population. Based on the morphological characters and the phylogenetic analyses, three groups, corresponding to each of the three Chattonella species, were roughly recognized. Nevertheless, this three-group distribution was insufficient to justify their distinction at the species rank. We therefore proposed a taxonomic revision with C. antiqua and C. ovata reduced to varieties of C. marina, this latter having name priority. We proposed a new status: C. marina var. antiqua (Hada) Demura & Kawachi and C. marina var. ovata (Y. Hara & Chihara) Demura & Kawachi.

Denaturing gradient gel electrophoresis shows that bacterial communities change with mid-ocean ballast water exchange.

Ships carry ballast water for better stability and to control trim. However, the discharge of ballast water near ports is known to transport invasive species from one coastal area to another. The exchange of ballast water on the high seas is supposed to reduce such invasions of exotic species. In this study, we used denaturing gradient gel electrophoresis (DGGE) to analyze the composition of the bacterial community in ballast water before and after such a mid-ocean exchange, and we also measured total bacterial counts. Our findings confirmed that the ballast water was replaced by the mid-ocean exchange, as indicated by the marked change in the composition of the bacterial community. There was also a significant decrease in bacterial abundance after the mid-ocean exchange. Finally, our findings support the incubation hypothesis, because the composition of the bacterial communities changed over time within the same ballast water.

Genetic structure of Japanese Chattonella marina (Raphidophyceae) populations revealed using microsatellite markers.

Chattonella marina var. antiqua and C. marina var. marina (Raphidophyceae) are red tide‐forming, harmful phytoplankton species. We investigated the genetic diversity and genetic relationship among the populations using microsatellite markers to identify putative sources of C. marina var. antiqua and C. marina var. marina in Japanese coastal populations. A positive correlation between genetic divergence and geographical distance (isolation by distance) was recognized for C. marina var. antiqua. The C. marina var. antiqua populations were established throughout a geological time scale, and genetic divergence had progressed in each population with gene flow depending on geographic distances. In contrast, isolation by distance was not observed for C. marina var. marina populations, and the genetic divergence among populations was extremely high. The Tokyo Bay population of C. marina var. marina, which was first recognized in 2008, had many private alleles but was related to the Kagoshima Bay population. The Tokyo Bay population may have been established by several invasions from the Kagoshima Bay population and other regions.

Life cycle of Chattonella marina (Raphidophyceae) inferred from analysis of microsatellite marker genotypes

Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single‐cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low‐nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.