Yoshikuni Hodoki

Bacteria biofilm encourages algal immigration onto substrata in lotic systems

I tested the effect of the density of attached bacteria on the amount of algal immigration in the early development of a periphyton community in an artificial stream by manipulating the density of the attached bacteria. Three densities were prepared by regulation of the incubation time. A suspension of algae was added to the stream, and the degree of algal attachment to substrata was compared among the treatments. Algal immigration was proportional to the density of attached bacteria on all substrata (glass, PVC, and slate), although density differed among substrata. Analysis of covariance (dependent variable, amount of attached algae; covariate, bacterial density) showed significant relationship between amounts of attached algae and bacterial densities, but did not show significant differences in the slopes and adjusted means among substrata. When acrylic beads were added with the suspension of attached algae, significant linear correlation was obtained between the amount of attached algae and the amount of acrylic beads on the substrata. Algal immigration was due to non-selective adsorption by attached bacterial biofilms on substrata, although the extent of bacterial colonization and biofilm formation may be affected by the substrata and other environmental factors (e.g., current conditions and water temperature).

Kinetoplastid flagellates overlooked by universal primers dominate in the oxygenated hypolimnion of Lake Biwa, Japan

Kinetoplastid flagellates, microscopically often detected from various aquatic environments and considered ubiquitous are seldom reported in molecular diversity studies with universal eukaryote DNA primers. To investigate this inconsistency, we examined nanoflagellate diversity in Lake Biwa, Japan by 18S rRNA gene clone libraries using universal eukaryote and kinetoplastid-specific primers. We also examined the abundance of kinetoplastids by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization. No, kinetoplastid sequences were detected in the universal eukaryote primers library from epilimnion and hypolimnion in different seasons. However, kinetoplastid flagellates were detected with kinetoplastid-specific probe from all of the seasons and contributed up to 11.9 and 36.0% of total eukaryotes in the epilimnion and hypolimnion, respectively. Thus, kinetoplastids probably are a significant, sometimes dominant, group in the hypolimnion, contributing up to 43.7% of the total flagellates. Using group-specific primers, kinetoplastid sequences were also obtained from both epilimnion and hypolimnion library. Therefore, we attributed the inconsistency to the divergent nature of 18S rRNA gene of kinetoplastids, which lead to their undetection in the universal eukaryote primer libraries. This study revealed that kinetoplastids have significant ecological importance in the hypolimnion of Lake Biwa, suggesting that these flagellates have been overlooked in other studies using universal eukaryote primers.

Effects of solar ultraviolet radiation on the periphyton community in lotic systems: comparison of attached algae and bacteria during their development

The effects of solar ultraviolet radiation (UVR) on the development of a periphyton community were studied in an outdoor artificial stream apparatus. Algal biomass, species composition, and bacterial cell density were measured under full sunlight and non-UVR (photosynthetically active radiation [PAR]-only) conditions. Attachment of algae was detected on days 6–9. Although the chlorophyll-a concentration under non-UVR conditions was 2–4 times that under full sunlight (PAR + UVR) throughout the experiment, neither net algal growth rate nor species composition differed significantly between the two light conditions. The relative carotenoid pigment contents of attached algae in the PAR + UVR condition were 1.1–1.3 times those in the non-UVR condition. Rates of increase of bacterial cell densities under the PAR + UVR condition were depressed by solar UVR for the first few days, although there were no apparent differences in the rates of increase between the light conditions later in the experiment. The small effect of UVR on the development of this periphyton community may be attributable to low UV flux at this study site and to the experimental conditions under which the algae were kept: a high physiological state with high nutrient conditions. Attached bacteria and algae that colonize substrata first are likely to be sensitive to solar UVR, and the negative effects of UVR are mitigated by the development of a periphyton community.

Detection of UVBR-sensitive and -tolerant bacteria in surface waters of the western North Pacific

In order to evaluate the effects of solar ultraviolet radiation (UVR) on eubacterial community composition, we examined the tolerance of eubacterial phylotypes to solar UV radiation in surface waters of the western North Pacific during September 2005. Bromodeoxyuridine (BrdU), a halogenated thymine analogue, was used for labeling newly synthesized DNA in proliferating cells. Thymine dimers (TD), which are specifically formed in DNA by biologically harmful ultraviolet B radiation (UVBR; 280-315nm), were also applied to detect UVB damaged genomes selectively. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) on the labeled samples revealed that UVBR-resistant cells showing active synthesis of DNA without accumulating TD, varied among phylotypes. In addition, UVBR-sensitive band positions with TD indicated inter-specific variations in sensitivity to UVBR. Phylogenetic analysis revealed that 12 DNA sequences were classified into eight phylogenetic groups: three Roseobacter, one Sphingomonas, two Gammaproteobacteria, one Actinobacteria, one Synechococcus, two Prochlorococcus, one plastid and one another group. A UVBR-resistant phylotype was affiliated to Erythrobacter sp. (previously designated as Sphingomonas sp.), which was distributed in warmer waters from the south of Oyashio to Kuroshio regions. A UVBR-sensitive phylotype was affiliated to Pseudoalteromonas sp. in Gammaproteobacteria. Dominant heterotrophic eubacteria were composed of both sensitive and resistant phylotypes. This is the first report on TD accumulated eubacterial phylotypes in oceanic surface waters.

Species-specific responses of freshwater diatoms to solar ultraviolet radiation

We studied species-specific growth responses of freshwater algae to solar ultraviolet radiation (UVR) by measuring growth-irradiance curves (G-E curves) of 17 algal diatoms (isolated from a periphyton community in a lotic system) in a solar simulator under photosynthetically available radiation plus UVR and minus UVR radiation condition. Algal growth responses to UVR were highly species-specific, and the photoinhibition parameter (β) of the +UVR condition differed greatly among species. We compared the degree of UVR effect on algal growth (UVR inhibition index: difference in between +UVR and -UVR conditions) with parameters derived from the G-E curve and with algal cell morphology. UVR inhibition indexes significantly correlated with the initial slope of light-limited portion of G-E curve (a) and with the irradiance at which the growth rate starts to saturate (E k ), but not with the cell surface to volume ratios. Low-light-adapted algae tended to be more susceptible to growth inhibition by UVR than high-light-adapted ones. Algae that formed dense colonies in the growth medium were relatively tolerant of UVR irrespective of E k values. Although past studies have emphasized that UVR effects on algae are algal-size dependent, the growth strategies dealing with solar radiation and algal growth forms appear to be more valid in correctly interpreting species-specific responses of algae to solar UVR.

Evaluation of Genetic Diversity in Zostera japonica (Aschers. et Graebn.) for Seagrass Conservation in Brackish Lower Reaches of the Hii River System, Japan

This study examined the genetic diversity and composition of 15 populations of Zostera japonica in the Hii River system, Japan. Genetic and genotypic diversity were consistently higher in populations in the Ohashi River than those in Lake Nakaumi, and the local tidal movements may explain the observed patterns of genetic diversity. Pairwise fixation index values among the populations in Lake Nakaumi were higher than among those in the Ohashi River, even though the ranges of the distances among the populations in the two locations were almost the same. The gene flow from the Ohashi River populations was important for some populations in Lake Nakaumi. The low genotypic diversity and gene flow in Lake Nakaumi seemed similar to those of marginal populations. If this low genotypic diversity and genetic differentiation were partly caused by human activities, the promotion of gene flow should be taken into account in conserving the populations in Lake Nakaumi.

Analysis of population clonal diversity using microsatellite markers in the salt marsh sedge Carex scabrifolia in western Japan

Carex scabrifolia, a perennial herb that commonly grows on sandbars in lagoons and tidal estuaries, is threatened by habitat reduction in some areas of Japan. Clonal diversity and the extent of gene flow among ten populations located along the Seto Inland Sea and the Japan Sea in western Japan were examined using six microsatellite loci. From 299 samples, we detected 77 multilocus genotypes. The mean number of alleles per population was 2.8 and the mean clonal diversity was 0.23. Many populations consisted of small patches, and the mean number of genets per patch was 2.0. The average number of alleles per locus and clonal diversity were positively correlated with the number of patches within a watershed. Gene flow was detected between the Ichikawa River and the Ohta River populations along the Seto Inland Sea, and weak differentiation among populations located along the Seto Inland Sea was observed. Our results suggest that effective conservation of C. scabrifolia populations should include maintaining all patches within a watershed regardless of population size, thus promoting genotype preservation.

Genotypic composition and the relationship between genotypic composition and geographical proximity of the cyanobacterium microcystis aeruginosa in western Japan

Microcystis aeruginosa is one of the bloom-forming harmful algae in freshwater ecosystems. We genetically characterized Microcystis populations during bloom-forming periods in various reservoirs, lakes, and ponds in Japan during 2009. Using phylogenetic analysis, we evaluated the relationship between current genotype expansions and geographic location within western Japan and intraspecific variation. Microcystis aeruginosa colonies were isolated at 15 sites and were analyzed by sequencing the 16S-23S internal transcribed spacer (ITS) region of the ribosomal operon, and the potential to produce toxins was assessed by PCR-based detection of the microcystin synthetase gene mcyG. In total, 171 colonies were separated into 41 genotypes. The highest genotypic composition was detected in the south basin of Lake Biwa and the lowest in Lagoon Iba. Cluster analysis indicated no obvious association between genotypic composition and geographic distance. Thus, clear genetic differentiation accompanied by geographic origins was not found in western Japan. The resulting neighbor-joining tree revealed 3 clusters, 2 of which contained strains that showed both nonamplification and amplification of the mcyG gene.

Direct and indirect effects of solar ultraviolet radiation on attached bacteria and algae in lotic systems

I examined the effects of solar ultraviolet radiation (UVR) on attached bacteria and algal densities in lotic systems in outdoor artificial stream apparatus. Flumes were covered with four types of film for UVR screening treatments, and attached bacterial cell densities and their temporal variations were compared between conditions excluding and including solar UVR. Attached bacterial cell densities were depressed by solar UVR, and both accrual rate and saturated density were significantly lower in the +UVR (full solar radiation) condition than in −UVR and dark conditions. Solar UVR also indirectly affected the rate of algal accrual. Microscopic direct observations of attached bacterial cell density and algae on substrata showed that solar UVR depressed the accrual of attached bacteria and consequently the frequency of sites with high bacterial cell density that can trap suspended algae in the water. The final amount of algal accrual in the +UVR condition was one-fourth of that in the −UVR condition. Therefore, the effects of solar UVR may be more serious in systems where periphyton are frequently removed by floods.

Adaptation of the Freshwater Bloom-Forming Cyanobacterium Microcystis aeruginosa to Brackish Water Is Driven by Recent Horizontal Transfer of Sucrose Genes

Microcystis aeruginosa is a bloom-forming cyanobacterium found in eutrophic water bodies worldwide. M. aeruginosa blooms usually occur in freshwater; however, they have also been reported to occur in brackish water. Because M. aeruginosa often produces the cyanotoxin microcystin, they are a major concern to public health and environment. Despite this, the ecology, genomic basis, and evolutionary process underlying the M. aeruginosa bloom invasion from fresh to brackish water have been poorly investigated. Hence, in the present study, we have sequenced and characterized genomes of two newly discovered salt-tolerant M. aeruginosa strains obtained from Japanese brackish water lakes (Lakes Shinji and Tofutsu). Both genomes contain a set of genes for the synthesis of osmolyte sucrose (sppA, spsA, and susA), hitherto identified in only one strain (PCC 7806) of M. aeruginosa. Chemical and gene expression analyses confirmed sucrose accumulation induced by salt. A comprehensive genetic survey of >200 strains indicated that sucrose genes are extremely rare in M. aeruginosa. Most surprisingly, comparative genome analyses of the three strains indicated extremely low genetic diversity in the sucrose genes compared with other core genome genes, suggesting very recent acquisitions via horizontal transfer. Invasion of M. aeruginosa blooms into brackish water may be a recent event triggered by anthropogenic eutrophication of brackish water.