Phylogenetic diversity of the picocyanobacterial community from a novel winter bloom in Lake Biwa

In Lake Biwa, picocyanobacteria blooms usually occur during the summer-autumn thermal stratification period. Intriguingly, a novel bloom was detected in winter 2015-2016, in which picocyanobacterial cell density increased by one order of magnitude despite lower water temperature, suggesting the possibility that "cold-water-preference" species dominate in the picocyanobacterial community. In the present study, we investigated the phylogenetic diversity of picocyanobacteria in Lake Biwa by analyzing the 16S rRNA gene. We found that the picocyanobacterial community were highly diverse in Lake Biwa, with eight Synechococcus-related operational taxonomic units (OTUs) detected in different seasons. These OTUs fell into distinct phylogenetic groups, and the majority were closely related to clusters reported previously. Notably, OTU04, detected during the winter bloom, was highly affiliated with sequences found in a variety of lakes, such as Tibetan lakes and Lake Superior, where the water bodies generally have a low trophic state and temperature, and different concentrations of total dissolved solids. Thus, we inferred that the group containing OTU04 may be a psychrotolerant lineage that is widely distributed in oligotrophic water systems with low-intermediate salinity

Influence of potential grazers on picocyanobacterial abundance in Lake Biwa revealed with empirical dynamic modeling

Picocyanobacteria in lakes generally occur as single cells (single-celled picocyanobacteria; SPcy) or colonies (colonial picocyanobacteria; CPcy), and the latter form has been considered an adaptation to grazing pressure. In addition to direct effects of grazing, grazers may also have important indirect effects on picocyanobacteria, such as those from nutrient regeneration and trophic cascades. Interactions between picocyanobacteria and their grazers in lakes can thus be complex and difficult to predict. To evaluate the influence of various grazers on SPcy and CPcy in Lake Biwa, Japan, we followed seasonal changes in their abundances and potential grazers at 2-week intervals over 2 years. The data collected were analyzed using empirical dynamic modeling (EDM), a model-free, nonlinear time-series method. We found that heterotrophic nanoflagellates (HNF), rotifers (Keratella, Polyarthra, and Trichocerca), cladocerans, and copepods played important and differing roles in controlling the abundances of SPcy and CPcy. Notably, HNF had an apparent positive influence on SPcy abundance, despite being considered major consumers of SPcy. This result suggested that the enhancement of SPcy growth due to nutrient regeneration by HNF might exceed losses from mortality due to grazing by HNF. EDM also suggested that colony formation by picocyanobacteria may be unidirectional, with SPcy tending to form CPcy. Our findings show that the seasonal dynamics of SPcy and CPcy in Lake Biwa are influenced by a variety of grazers, which may play differing ecological roles in the aquatic food web

A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and CARD-FISH. We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to marine isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single cell genomes of marine diplonemids shows they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone

Seasonal dominance of CL500-11 bacterioplankton (phylum Chloroflexi) in the oxygenated hypolimnion of Lake Biwa, Japan

Uncultured bacteria affiliated with the CL500-11 cluster (phylum Chloroflexi) were first reported from the oxygenated hypolimnion of Crater Lake (USA) as a predominant bacterioplankton, although this dominance has not been reported in other environments. In this study, we showed that CL500-11 is also dominant in the oxygenated hypolimnion of Lake Biwa (Japan) and followed its spatiotemporal succession using fluorescent in situ hybridization. CL500-11 cells were almost absent [ 10% of DAPI-stained cells; maximum = 16.5%) in the hypolimnion during the stratification period, and decreased to below the detection limit with the collapse of the thermocline. This pattern was observed over two annual cycles. A longitudinal assessment also showed that CL500-11 was the dominant bacterium in the hypolimnion over the whole lake, but was generally undetectable in the stratified epilimnion. These data suggest that CL500-11 is acclimated to the oxygenated hypolimnion and is a potentially important component of the pelagic biogeochemical cycling of the lake. A comparative analysis of 16S rRNA gene sequences revealed that almost all CL500-11 sequences previously deposited in the database were detected from hypolimnion or holomictic water in deep oxic freshwater lakes, suggesting that the bacteria may form one of the common lineages residing in an aerobic hypolimnetic niche

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; 280-315 nm), 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 8 phylogenetic groups: 3 Rosecbacter, 1 Sphingomonas, 2 Gammaproteobacteria, 1 Actinobacteria, 1 Synechococcus, 2 Prochlorococcus, 1 plastid and 1 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

Tempo-spatial patterns of bacterial community composition in the western North Pacific Ocean

In the western North Pacific, where subarctic Oyashio waters encounter subtropical Kuroshio waters, phylotype composition of heterotrophic bacteria was estimated by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S ribosomal DNA. Total bacterial abundance was also measured by flow cytometry. The study area was divided into four water masses: coastal, Oyashio, Kuroshio, and the Kuroshio-Oyashio transition. Abundances of heterotrophic bacteria in the Oyashio, Kuroshio, and Kuroshio-Oyashio transition regions ranged from 0.2 x 10^[5] cells mL^[-1] to 1.4 x 10^[5] cells mL^[-1] and were not significantly different, except in the Oyashio region during September. Bacterial compositions were distinct in each water mass. Furthermore, phylotype distributions differed between surface and subsurface waters in the Kuroshio-Oyashio transition region. Out of 61 DGGE bands obtained, 41 were successfully identified as 31 phylotypes: 22 Gammaproteobacteria, 4 Alphaproteobacteria, 2 unknown bacteria, 2 cyanobacteria, and 1 plastid. Although the Gammaproteobacteria OM60 clade was eurytopic in the study area, Psychrobacter glacincola and the uncultured Gammaproteobacteria SAR92 clade were often observed in the Oyashio region. Overall, our results indicated that Gammaproteobacteria were predominant in the bacterial community, which was influenced by the hydrographic properties of each water mass in the study area

Detection and identification of potentially toxic cyanobacteria: Ubiquitous distribution of Microcystis aeruginosa and Cuspidothrix issatschenkoi in Japanese lakes

We studied the frequency and composition of potentially toxic cyanobacteria in 30 samples from 25 Japanese lakes using automated rRNA intergenic spacer analysis (ARISA) and eight primer sets for nodularin, microcystin, cylindrospermopsin, anatoxin-a, and saxitoxin synthetase genes. Potential microcystin- and anatoxin-a-producers were detected in 25 and 7 samples, respectively. Cylindrospermopsin- or saxitoxin-producers were not detected. PCR and clone library analyses indicated that Microcystis was the sole microcystin-producing genus. Moreover, potential microcystin-producing Microcystis strains were detected in 25 of 26 samples which included Microcystis ARISA fragments, suggesting that toxic Microcystis is ubiquitous. Potential anatoxin-a-producers detected in the samples were estimated to be Cuspidothrix issatschenkoi (synonym: Aphanizomenon issatschenkoi) from clone library analyses of the anatoxin-a biosynthesis gene cluster and 16S–23S rDNA intergenic spacer region. Anatoxin-a-producing strains of C. issatschenkoi are known from two lakes in New Zealand and Germany. In contrast, C. issatschenkoi was the second most common toxic cyanobacteria in our study. This is the first report of potential anatoxin-a-producing C. issatschenkoi strains in Asia

Grazing impact on the cyanobacterium Microcystis aeruginosa by the heterotrophic flagellate Collodictyon triciliatum in an experimental pond

We estimated the grazing impact of the heterotrophic flagellate Collodictyon triciliatum on the harmful, bloom-forming cyanobacterium Microcystis aeruginosa in an experimental pond during a Microcystis bloom from summer to winter in 2010. For these experiments, we calculated the grazing rates from the digestion rate of C. triciliatum and its food vacuole contents. During the study period, M. aeruginosa exhibited one bloom event with a maximum density of 1.1 × 10[5] cells ml[−1]. The cell density of C. triciliatum fluctuated from below the detection limit to 291 cells ml[−1]. The number of M. aeruginosa cells ingested by C. triciliatum food vacuoles ranged between 0.4 and 10.8 cells flagellate[−1], and the digestion rate of C. triciliatum at 25 °C was 0.73 % cell contents min[−1]. The grazing rate of C. triciliatum on the M. aeruginosa prey was 0.2–6.9 cells flagellate[−1] h[−1], and its grazing impact was 0.0–25.3 % standing stock day[−1]. The functional response of C. triciliatum to the M. aeruginosa prey followed the Michaelis–Menten model of significance (r[2] = 0.873, p < 0.001) in our experimental systems, in which the prey concentration varied from 1.0 × 10[4] to 2.1 × 10[6] cells ml[−1]. The maximum grazing rate was 6.2 prey cells grazer[−1] h[−1], and the half-saturation constant was 1.2 × 10[5] cells ml[−1]. We present evidence that C. triciliatum grazing explained the remarkable decrease in M. aeruginosa cell density in the pond. The present study is the first demonstration of the high potential of protistan grazing on M. aeruginosa to reduce cyanobacterial blooms

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

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