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.

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.

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.

Radioactive cesium dynamics derived from hydrographic observations in the Abukuma River Estuary, Japan.

Large quantities of radioactive materials were released into the air and the ocean as a result of the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, caused by the 2011 Tohoku earthquake and the subsequent major tsunami off the Pacific coast. There is much concern about radioactive contamination in both the watershed of the Abukuma River, which flows through Fukushima Prefecture, and its estuary, where it discharges into the sea in Miyagi Prefecture. We investigated radioactive cesium dynamics using mixing diagrams obtained from hydrographic observations of the Abukuma River Estuary. Particulate radioactive cesium dominates the cesium load in the river, whereas the dissolved form dominates in the sea. As the salinity increased from <0.1 to 0.1-2.3, the mixing diagram showed that dissolved radioactive cesium concentrations increased, because of desorption. Desorption from suspended particles explained 36% of dissolved radioactive cesium in estuarine water. However, the dissolved and particulate radioactive cesium concentrations in the sea decreased sharply because of dilution. It is thought that more than 80% of the discharged particulate radioactive cesium was deposited off the river mouth, where the radioactive cesium concentrations in sediment were relatively high (217-2440 Bq kg(-1)). Radioactive cesium that was discharged to the sea was transported southward by currents driven by the density distribution.