Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences.

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Subarctic-scale transport of 134Cs to ocean surface off northeastern Japan in 2020.

We studied the spatiotemporal variations in 134Cs, 137Cs, and 228Ra concentrations at the sea surface off southeastern Hokkaido, Japan (off-Doto region) from 2018 to 2022 using low-background γ-spectrometry. The 134Cs concentrations in the off-Doto region, decay-corrected to the date of the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, exhibited wide lateral variation each year (e.g., 0.7-1.1 mBq/L in 2020). By studying the 228Ra concentrations and salinity, this variation was explained based on the current mixing patterns. Furthermore, the 134Cs concentrations in the waters highly affected by the Oyashio Current (OYC) gradually increased from 2018 to 2020, and subsequently decreased in 2022. This implies that the water mass maximally contaminated with 134Cs was transported back to the side of the Japanese islands 10 years after the FDNPP accident along with counter-clockwise currents (e.g., the OYC) in the northern North Pacific Ocean. The 134Cs concentrations in the OYC-affected waters in the off-Doto region in 2020 were ~ 1/6 times those in the 134Cs-enriched core of waters off the western American Coast in 2015, which can be ascribed to dilution via spatial dispersion during subarctic current circulation. Overall, we elucidated the ocean-scale subarctic current systems in the northwestern North Pacific Ocean, including water circulation timespans.

Empirical estimation of marine phytoplankton assemblages in coastal and offshore areas using an in situ multi-wavelength excitation fluorometer.

Phytoplankton assemblages are essential for understanding the quality of primary production in marine ecosystems. Here, we describe the development of a methodology for monitoring marine phytoplankton assemblages using an in situ multi-wavelength excitation fluorometer (MEX). The MEX recorded the fluorescence excited with nine light-emitting diodes, temperature, and sensor depth. We prepared reference datasets comprising MEX fluorescence and plant pigment-based phytoplankton assemblages of nine chemotaxonomy groups (diatoms, dinoflagellates, cryptophytes, chlorophytes, haptophytes type 3, haptophytes type 4, prasinophytes, cyanophytes, and prochlorophytes). Conversions from the MEX fluorescence to the phytoplankton assemblages were conducted with two processes. First, target MEX fluorescence was decomposed using a linear inverse model for calculating coefficients. Second, pigment-based chemotaxonomy of the target MEX fluorescence was reconstructed using the coefficients and the chemotaxonomy assemblages of the reference data. Cross-validation analyses indicated good estimation of the proportion of diatoms, dinoflagellates, cryptophytes, cyanophytes, and prochlorophytes with MEX, and when chlorophytes, haptophytes and prasinophytes were summarized as other eukaryotes, the positive correlation was seen between proportions estimated with MEX and pigments as same as other five chemotaxonomy groups. Repeated MEX observations were conducted in the Kuroshio, the Sea of Japan, the Oyashio, and the Okhotsk Sea. The water-column integrated biomass indicated that the diatoms were an important primary producer in the Oyashio and the Okhotsk Sea, while eukaryotes were important in the Sea of Japan and prochlorophytes were important in the Kuroshio. Our method with the MEX will be a powerful tool to understand and estimate the chemotaxonomy-level assemblages and biomass in the ocean.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Micro-size plankton abundance and assemblages in the western North Pacific Subtropical Gyre under microscopic observation.

While primary productivity in the oligotrophic North Pacific Subtropical Gyre (NPSG) is changing, the micro-size plankton community has not been evaluated in the last 4 decades, prompting a re-evaluation. We collected samples over three years (2016-2018) from depths of 10 to 200 m (n = 127), and the micro-size plankton were identified and counted to understand the heterogeneity of micro-size plankton community structure. The assemblages were consistent to the those of 4 decades ago. Dinophyceae (dinoflagellates) were the most numerically abundant, followed by Cryptophyceae and Bacillariophyceae (diatoms). The other micro-size plankton classes (Cyanophyceae, Haptophyceae, Dictyochophyceae, Euglenophyceae, and Prasinophyceae) were not always detected, whereas only Trichodesmium spp. was counted in the Cyanophyceae. Other unidentified autotrophic and heterotrophic flagellates were also significantly present, and their numeric abundance was higher than or at the same level as was that of the Dinophyceae. In the Dinophyceae, Gymnodiniaceae and Peridiniales were abundant. The chlorophyll a concentration and these class-level assemblages suggested micro-size plankton is not a major primary producer in this area. We applied generalized additive models (GAMs) and principal coordination analyses (PCoAs) to evaluate the habitats of every plankton group and the heterogeneity of the assemblages. The GAMs suggested that every classified plankton abundance showed a similar response to salinity, and we observed differences in habitats in terms of temperature and nitrate concentrations. Based on the PCoAs, we observed unique communities at the 200 m depth layer compared with those at the other sampling layers. The site scores of PCoAs indicated that the micro-size plankton assemblages are most heterogeneous at the 10 m depth layer. At such depth, diazotrophic Cyanophyceae (Trichodesmium spp.) are abundant, particularly in less-saline water. Therefore, nitrogen fixation may contribute to the heterogeneity in the abundance and assemblages in the western NPSG.

18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific.

Eel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.

A Preliminary Metagenome Analysis Based on a Combination of Protein Domains.

Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions.

Seasonal and geographical distribution of near-surface small photosynthetic eukaryotes in the western North Pacific determined by pyrosequencing of 18S rDNA.

In this study, we investigated the distribution of small photosynthetic eukaryotes in the near-surface layer of the western North Pacific at four stations, including two oceanic stations where the subarctic Oyashio and subtropical Kuroshio currents influence a transition region and the bay mouth and head of the Sendai Bay, from April 2012 to May 2013. Flow cytometry was applied to sort small photosynthetic eukaryotes (<5 µm), and high-throughput sequencing of 18S rDNA was performed. Our taxonomic analysis showed that 19/195 operational taxonomic units (OTUs) were frequently distributed among all sites. Composition analysis showed that the OTUs had characteristic patterns and were divided into four main groups. Two groups reflected the low-saline water and winter season, with the characteristic OTUs belonging to diatoms; Chaetoceros and Leptocylindrus were characteristic of low saline water, and two diatom genera (Minidiscus and Minutocellus) and Cryptomonadales-related OTUs were prevalent in the winter. Our results indicate that the community composition of small photosynthetic eukaryotes seasonally changes in a dynamic manner according to variations in water properties.

Spatial variation of bacterial community composition at the expiry of spring phytoplankton bloom in Sendai Bay, Japan.

In order to characterize how bacterial communities are propagated over spatial scales in a coastal area, the bacterial community composition was examined along with a transect line set in a bay at an expiry of spring phytoplankton bloom. Four distinctive bacterial communities were found within the bay by a fingerprinting method of 16S rRNA gene amplicons. The most widely distributed one was distributed in the surface and middle layers at whole area of the bay. The water was characterized by low inorganic nutrients concentration and high bacterial abundance, suggesting that the bacterial community had been developed in the bloom. Pyrosequencing analyses of the gene amplicons indicated that Rhodobacteriaceae and Flavobacteriaceae were abundant in the bacterial community, though the most abundant bacterial taxon was SAR11. The second group was distributed in the bottom water at the coastal side of the bay where considerably high Chl. a concentration was observed, probably because of the sedimentation of phytoplankton bloom. The community diversity was high and Alteromonadaceae, Saprospiraceae, and some families of Actinobacter existed more in this community than the others. The third group was distributed in the deep water near the border with the outside of the bay. The ratio of SAR11 was the highest in this community; besides, Burkholderianceae and Rhodospilliraceae existed in relatively high abundances. Another bacterial community having intermediate characters was observed in the middle to bottom layers around a central part of the bay where vertical water mixing was observed. These findings suggest that spatially different bacterial communities were formed under the influences of phytoplankton bloom and/or hydrographic events such as oceanic seawater intrusion of the bay.

Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean.

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 µm in width and 4.0-6.0 µm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 µmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.

Diazotrophy under continuous light in a marine unicellular diazotrophic cyanobacterium, Gloeothece sp. 68DGA.

Nitrogenase is extremely sensitive to molecular oxygen (O(2)), and unicellular diazotrophic cyanobacteria separate nitrogen (N(2))-fixation and photosynthesis to protect nitrogenase from O(2) produced by photosynthesis. When grown under 12 h light/12 h dark cycles (LD), the marine unicellular diazotrophic cyanobacterium Gloeothece sp. 68DGA expressed the nitrogenase protein and its activity (acetylene reduction activity) only during the dark phase. However, this strain was able to grow diazotrophically under continuous light (CL). To determine whether nitrogenase synthesis and N(2)-fixation are temporally separated from photosynthesis in the Gloeothece cells that have fully acclimated to CL, the proportion of cells containing nitrogenase (the Fe-protein of nitrogenase) in the culture was measured using an immunocytochemical technique. Cells were grown in a continuous-culture device to maintain constant cell density. Under LD, the cells showed diurnal oscillation of nitrogenase activity, photosynthesis, respiration and the expression and the abundance of the Fe-protein. The oscillation was gradually reduced after the transfer of the cells to CL, and was lost after 23-25 days of cultivation under CL. In CL-acclimated cultures, the Fe-protein was always detected in about 94 % of the cells, although the nitrogenase activity was about one-third of the maximum activity in LD-acclimated cultures. These results suggest that synthesis of nitrogenase proceeds without diurnal oscillation in the CL-acclimated cells of Gloeothece sp. 68DGA. As the respiration rate in CL-acclimated culture was as high as the maximum rate observed in LD-acclimated culture, O(2)-uptake mechanism(s) may have been upregulated to maintain low intracellular pO(2).

Stable top-up operation at SPring-8.

Top-up operation allows SPring-8 to provide highly stable X-ray beams with arbitrary filling patterns. The implementation of top-up operation is described, with a focus on the simultaneous achievement of stability of stored current, beam orbit, purity of an isolated single bunch, and beam injection efficiency. Stored-current fluctuations have been routinely reduced to a level of 10(-3). Stored-beam oscillation on frequent beam injection, which was originally regarded as the most serious problem, has been successfully suppressed to a sufficiently low level that it never perturbs imaging experiments. Current impurities in nominally empty buckets have been reduced to a level of 10(-9) over more than one week of operation, making possible the measurement of time-resolved spectra using high-current bunches. Finally, excellent injection efficiency, higher than 80%, is routinely obtained, even for small undulator gaps, which is critical for preventing radiation damage to insertion-device magnets and to reduce leakage radiation. The process of achieving highly stabilized top-up operation at SPring-8 and its utility for user experiments are described.