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.

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.

Toxic HAB species from the Sea of Okhotsk detected by a metagenetic approach, seasonality and environmental drivers.

During recent decades, the distribution of harmful algal bloom (HAB) species has expanded worldwide together with the increase of blooms and toxicity events. In this study, the presence of toxic HAB species in the Sea of Okhotsk was investigated based on metagenetic data collected during 6 years of weekly monitoring. Operational taxonomic units (OTUs) associated with the toxic HAB species were detected based on amplifying 18S V7-V9 and 28S D1 rRNA gene regions. In total, 43 unique OTUs associated with toxic HAB species were revealed, with 26 of those previously not reported from the Sea of Okhotsk. More OTUs belonging to dinoflagellates were detected by 18S, whereas a similar number of OTUs associated with dinoflagellates and diatoms were detected by targeting the 28S region. Species belonging to genera Alexandrium, Karenia and Karlodinium were mainly associated with OTUs under Dinophyceae, whereas Bacillariophyceae was represented by the species belonging to genus Pseudo-nitzschia. From the detected OTUs, 22 showed a clear seasonal pattern with the majority of those appearing during summer-autumn. For Alexandrium pacificum, Aureococcus anophagefferens, and Pseudo-nitzschia pungens, the seasonal pattern was detected based on both rRNA regions. Additionally, 14 OTUs were detected during all seasons and two OTUs appeared sporadically. OTUs associated with the toxic species had low relative read abundances, which together with other factors such as similar and variable morphology as well as usage of fixatives, may explain why those species have previously not been detected by light microscopy. Environmental parameters, especially water temperature, significantly (<0.05) influenced the variability in OTU relative abundances and displayed significant (<0.05) correlations with the unique OTUs. The results of this study demonstrate the usefulness of the metagenetic approach for phytoplankton monitoring, which is especially relevant for detecting toxic HAB species.

Detection and activity of iodine-131 in brown algae collected in the Japanese coastal areas.

Iodine-131 (physical half-life: 8.04 days) was detected in brown algae collected off the Japanese coast. Brown algae have been extensively used as bioindicators for radioiodine because of their ability to accumulate radionuclides in high concentration factors. The maximum measured specific activity of (131)I in brown algae was 0.37 + or - 0.010 Bq/kg-wet. Cesium-137 was also detected in all brown algal samples used in this study. There was no correlation between specific activities of (131)I and (137)Cs in these seaweeds. The specific activity of (137)Cs ranged from 0.0034 + or - 0.00075 to 0.090 + or - 0.014 Bq/kg-wet. Low specific activity and minimal variability of (137)Cs in brown algae indicated that past nuclear weapon tests were the source of (137)Cs. Although nuclear power stations and nuclear fuel reprocessing plants are known to be pollution sources of (131)I, there was no relationship between the sites where (131)I was detected and the locations of nuclear power facilities. Most of the sites where (131)I was detected were near big cities with large populations. Iodine-131 is frequently used in diagnostic and therapeutic nuclear medicine. On the basis of the results, we suggest that the likely pollution source of (131)I, detected in brown seaweeds, is not nuclear power facilities, but nuclear medicine procedures.

Detection and temporal variation of (60)Co in the digestive glands of the common octopus, Octopus vulgaris, in the East China Sea.

(60)Co were detected in common octopus specimens collected in the East China Sea in 1996-2005. The source of (60)Co has remained unclear yet. Stable isotope analyses showed that there was no difference in stable Co concentrations between octopus samples with (60)Co and without (60)Co. This result showed that the stable Co in the digestive gland of octopus potentially did not include a trace amount of (60)Co and the source of (60)Co existed independently. Furthermore, investigations of octopus in other area and other species indicated that the origin of the source of (60)Co occurred locally in the restricted area in the East China Sea and not in the coastal area of Japan. Concentrations of (60)Co have annually decreased with shorter half-life than the physical half-life. This decrease tendency suggests that the sources of (60)Co were identical and were temporary dumped into the East China Sea as a solid waste.

Temporal variations of 90Sr and 137Cs concentrations and the 137Cs/90Sr activity ratio in marine brown algae, Undaria pinnatifida and Laminaria longissima, collected in coastal areas of Japan.

The anthropogenic radionuclides, 90Sr and 137Cs, were measured in two marine algal species, wakame seaweed (Undaria pinnatifida) and edible kelp (Laminaria longissima), collected in four coastal areas of Japan during 1998-2008. Although 90Sr and 137Cs could be detected at all sampling sites, the concentrations of 90Sr and 137Cs were at low levels and those in some samples were below the detection limit. These low concentrations and the small variation of both concentrations and the 137Cs/90Sr activity ratio indicate that the source of 90Sr and 137Cs detected in this study originated from the global fallout deposition following atmospheric nuclear-bomb tests in the past. There were no significant differences in both concentrations of 90Sr and 137Cs in wakame seaweed among three sampling sites. Although wakame seaweed is extensively distributed in southern and central Japan, it does not occur in northern areas and so edible kelp was monitored. The concentrations of 90Sr and 137Cs in edible kelp were significantly different from those in wakame seaweed in some sampling sites. These differences could be due to the difference in the concentrations of 90Sr and 137Cs in the surrounding seawater or the difference in species. The combined data with data from the previous report and the preexisting database showed that wakame seaweed incorporated 137Cs through a different pathway from that of 90Sr. The combined data also suggested that wakame seaweed responded differently to the source of 137Cs.

Recent comparability of oceanographic nutrients data: results of a 2003 intercomparison exercise using reference materials.

An intercomparison exercise was conducted using the recently developed Reference Material for Nutrients in Seawater (RMNS). Discrepancies of reported values among laboratories were greater than the homogeneity of RMNS samples and the reported analytical precision of nutrients. The variability of in-house standards of the participating laboratories might be the most likely source of interlaboratory discrepancies. Therefore, the use of common reference materials, i.e. certified RM, is essential to establish and improve the comparability of nutrient data of the world's oceans.