DNA metabarcoding focused on difficult-to-culture protists: An effective approach to clarify biological interactions.

DNA metabarcoding on a single organism is a promising approach to clarify the biological interactions (e.g., predator-prey relationships and symbiosis, including parasitism) of difficult-to-culture protists. To evaluate the effectiveness of this method, Radiolaria and Phaeodaria, which are ecologically important protistan groups, were chosen as target taxa. DNA metabarcoding on a single organism focused on the V9 region of the 18S rRNA gene revealed potential symbionts, parasites and food sources of Radiolaria and Phaeodaria. Previously reported hosts and symbionts (parasites) were detected, and newly recognized combinations were also identified. The contained organisms largely differed between Radiolaria and Phaeodaria. In Radiolaria, members of the same order tended to contain similar organisms, and the taxonomic composition of possible symbionts, parasites, and food sources was fixed at the species level. Members of the same phaeodarian family, however, did not contain similar organisms, and body part (i.e., the central capsule or the phaeodium) was the most important factor that divided the taxonomic composition of detected organisms, implying that the selection of appropriate body part is important when trying to ascertain contained organisms, even for unicellular zooplankton. Our results show that DNA metabarcoding on a single organism is effective in revealing the biological interactions of difficult-to-culture protists.

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.

Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454.

Metagenetics is a rapid and taxonomically comprehensive method for revealing community structures within environmental samples, based on large amounts of sequence data produced by high-throughput sequencers. Because community structures of planktonic copepods are important in the ocean owing to their high diversity and abundance, a metagenetic analysis of the 28S D2 region using Roche 454 was previously developed. However, the Illumina MiSeq platform with a high sequence output is being used more frequently in metagenetics, and non-calanoid copepods have not previously been fully evaluated. Here, we evaluated an Illumina MiSeq-based metagenetic analysis using a mock community and field-collected samples that were examined in a previous study using Roche 454, and the community structure, including non-calanoid copepods, was compared among morphological and metagenetic analyses. We removed a singleton read and applied an appropriate abundance threshold to remove erroneous Molecular Operational Taxonomic Units (MOTUs) with low-abundance sequences in the MiSeq-based analysis. Results showed that the copepod community was successfully characterized using Illumina MiSeq. Higher-quality sequences were obtained using MiSeq than by Roche 454, which reduced the overestimation of diversity, especially at a strict 99% similarity threshold for MOTU clustering. Taxonomic compositions in terms of both biomass and presence/absence of species, including non-calanoids, were more appropriately represented in the MiSeq- than in Roche 454-based analysis. Our data showed that metagenetic analysis using Illumina MiSeq is more useful for revealing copepod communities than Roche 454, owing to the lower cost and higher quality.

Southwest intrusion of 134Cs and 137Cs derived from the Fukushima Dai-ichi nuclear power plant accident in the Western North Pacific.

Enormous quantities of radionuclides were released into the ocean via both atmospheric deposition and direct release as a result of the Fukushima Dai-ichi Nuclear Power Plant (FNPP) accident. This study discusses the southward dispersion of FNPP-derived radioactive cesium (Cs) in subsurface waters. The southernmost point where we found the FNPP-derived (134)Cs (1.5-6.8 Bq m(-3)) was 18 °N, 135 °E, in September 2012. The potential density at the subsurface peaks of (134)Cs (100-500 m) and the increased water column inventories of (137)Cs between 0 and 500 m after the winter of 2011-2012 suggested that the main water mass containing FNPP-derived radioactive Cs was the North Pacific Subtropical Mode Water (NPSTMW), formed as a result of winter convection. We estimated the amount of (134)Cs in core waters of the western part of the NPSTMW to be 0.99 PBq (decay-corrected on 11 March 2011). This accounts for 9.0% of the (134)Cs released from the FNPP, with our estimation revealing that a considerable amount of FNPP-derived radioactive Cs has been transported to the subtropical region by the formation and circulation of the mode water.