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.

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.

Correlation of proline, hydroxyproline and serine content, denaturation temperature and circular dichroism analysis of type I collagen with the physiological temperature of marine teleosts.

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.

Intrusion of Fukushima-derived radiocaesium into subsurface water due to formation of mode waters in the North Pacific.

The Fukushima Dai-ichi Nuclear Power Plant accident in March 2011 released radiocaesium ((137)Cs and (134)Cs) into the North Pacific Ocean. Meridional transects of the vertical distribution of radiocaesium in seawater were measured along 147 °E and 155 °E in October-November 2012, 19 months after the accident. These measurements revealed subsurface peaks in radiocaesium concentrations at locations corresponding to two mode waters, Subtropical Mode Water and Central Mode Water. Mode water is a layer of almost vertically homogeneous water found over a large geographical area. Here we show that repeated formation of mode water during the two winter seasons after the Fukushima accident and subsequent outcropping into surface water transported radiocaesium downward and southward to subtropical regions of the North Pacific. The total amount of Fukushima-derived (134)Cs within Subtropical Mode Water, decay-corrected to April 2011, was estimated to be 4.2 ± 1.1 PBq in October-November 2012. This amount of (134)Cs corresponds to 22-28% of the total amount of (134)Cs released to the Pacific Ocean.

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.

Use of otolith for detecting strontium-90 in fish from the harbor of Fukushima Dai-ichi Nuclear Power Plant.

To clarify the level of contamination with radioactive cesium (radiocesium) discharged from Fukushima Dai-ichi Nuclear Power Plant (FDNPP), three fish species caught in the main harbor of FDNPP were subjected to γ-ray analysis. The concentration of radiocesium in muscle differed among individual fish, even those of similar size of the same species, and showed little relation to the standard length of fish. The maximum concentration of radiocesium (202 kBq/kg wet) was detected from fat greenling samples. A comparison to data from outside the port indicated that the level of radiocesium contamination inside the port was higher than that outside. We found that ß-rays were emitted from otoliths of fishes caught in the port of FDNPP. ß-ray intensities were correlated with the concentrations of radiocesium in muscles of the three fish species. In Japanese rockfish, the ß-ray count rates from otoliths were significantly correlated with the concentration of radiocesium and (90)Sr in the whole body without internal organs of Japanese rockfish. However, no ß-rays were detected from brown hakeling samples collected around FDNPP, suggesting that the detection of ß-rays from otoliths may indicate living in the main harbor of FDNPP.

Exposure of a herbivorous fish to ¹³4Cs and ¹³7Cs from the riverbed following the Fukushima disaster.

Ayu Plecoglossus altivelis, a herbivorous fish, is an important fishery resource and key component of the foodweb in many Japanese streams. Radionuclide contamination of this species is likely transferred to higher trophic levels, include humans, in the food chain. After the Fukushima accident in March 2011, ayu were exposed to highly contaminated silt while feeding on algae attached to the riverbed stones. To understand the route by which herbivorous fish are exposed to radionuclides, the activity concentrations of sum of (134)Cs and (137)Cs (radiocesium) were analyzed in riverbed samples (algae and silt) and in the internal organs and the muscle of ayu in five river systems in the Fukushima Prefecture between summer 2011 and autumn 2013. Although there was a positive correlation between the radiocesium activity concentrations in the muscle and the internal organs of ayu, the median activity concentration in the muscle was much lower than those in the internal organs. The activity concentrations of radiocesium in the riverbed samples and the internal organs and the muscle of ayu were correlated with contamination levels in soil samples taken from the watershed upstream of the sample sites. The results of the generalized linear mixed models suggest that the activity concentrations in both the internal organs and the muscle of ayu declined over time. Additionally, the activity concentrations in the internal organs were correlated with those in the riverbed samples that were collected around the same time as the ayu. The activity concentrations in the muscle were correlated with ayu body size. Our results suggest that ayu ingest (134)Cs and (137)Cs while grazing silt and algae from the riverbed, and a part of the (134)Cs and (137)Cs is assimilated into the muscle of the fish.

Radiocesium contamination of greenlings (Hexagrammos otakii) off the coast of Fukushima.

We measured the radiocesium ((134)Cs and (137)Cs) contamination of 236 greenlings (Hexagrammos otakii) off the coast of Fukushima Prefecture in Japan, following the accident at the Fukushima Daiichi Nuclear Power Plant in March 2011. The radiocesium concentrations of greenlings caught approximately 40 km south of the power plant were significantly higher than those of greenlings caught approximately 50 km north of the power plant. The radiocesium concentrations of greenlings caught in southern waters were significantly higher in shallow than in deep waters. Meanwhile, two outlier specimens of greenlings with higher (137)Cs concentrations, 16,000 Bq/kg-wet on 1 August 2012 and 1,150 Bq/kg-wet on 8 May 2013, were caught approximately 20 km from the power plant. Our calculations suggest that the probability of two such outlier specimens being found off the coast of Fukushima is exceedingly low. By contrast, extremely contaminated greenlings were frequently caught in the power plant port (geometric mean of (137)Cs = 17,364 Bq/kg-wet). Our results suggest that the two outlier greenlings with higher (137)Cs concentrations migrated from the power plant port. Continued close monitoring of radiocesium concentrations in the area should be done to ensure the safety of food supplies.

Five-minute resolved spatial distribution of radiocesium in sea sediment derived from the Fukushima Dai-ichi Nuclear Power Plant.

The spatial distributions of radiocesium concentration in sea sediment to a core depth of 14 cm were investigated in the offshore region from the Fukushima Prefecture to the northern part of the Ibaraki Prefecture in February and July 2012, at a spatial resolution of 5 min of latitude and longitude. The concentrations in the area south of the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) were generally higher than those in the area north of it. In the southern area, a band of especially high concentration with a width about 20 km was present in the region shallower than 100 m, and a narrow minimal concentration band was found along the 200-m isobaths. In more than half of all cases, the vertical core profiles of radiocesium concentration generally showed an exponential decreasing trend with depth. However, in the area north of the FDNPP, where the radiocesium concentrations tended to be very low, radiocesium concentrations that had similar or larger magnitude compared with those of the most-surface layer were often found in deeper layers. Relatively good correlations were found between radiocesium concentrations and grain sizes of the most-surface sediment. The vertical profile of radiocesium concentration also had a relationship with grain size. In other case, the radiocesium concentration in the sediment seems to have had a dependence on the radiocesium concentration in bottom seawater, suggesting that the quantity of radiocesium supplied and the grain size were major factors determining the spatial distribution pattern of the radiocesium concentration after the FDNPP accident.

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.

Distribution of local 137Cs anomalies on the seafloor near the Fukushima Dai-ichi Nuclear Power Plant.

An estimated 3.5±0.7×10(15) Bq of (137)Cs is thought to have been discharged into the ocean following the melt down at Fukushima Dai-ichi Nuclear Power Plant (F1NPP). While efforts have been made to monitor seafloor radiation levels, the sampling techniques used cannot capture the continuous distribution of radionuclides. In this work, we apply in situ measurement techniques using a towed gamma ray spectrometer to map the continuous distribution of (137)Cs on the seafloor within 20 km of the F1NPP. The results reveal the existence of local (137)Cs anomalies, with levels of (137)Cs an order of magnitude higher than the surrounding seafloors. The sizes of the anomalies mapped in this work range from a few meters to a few hundreds of meters in length, and it is demonstrated that the distribution of these anomalies is strongly influenced by meter scale features of the terrain.

Population subdivision of Japanese flounder Paralichthys olivaceus in the Pacific Coast of Tohoku Japan detected by means of mitochondrial phylogenetic information.

This study deals with mitochondrial phylogenetic information of Japanese flounder in the Pacific coast of Tohoku Japan to estimate the genetic population subdivision that was undetectable by conventional population statistics. We determined complete sequences of mitochondrial NADH dehydrogenase subunit-2 (ND2) and subunit-5 (ND5) genes for 151 individuals from northern (Aomori and Iwate prefectures, 40-41°N) and southern (Miyagi and Fukushima prefectures, 37-38°N) waters. Samples from both waters showed high genetic diversity, including 126 haplotypes. These haplotypes were located at mixed and nested positions on an inferred phylogenetic tree, and traditional F-statistics indicated no significant population divergence (φ(ST) = -0.00335, p > 0.05), corroborating our previous study. Three variable sites, however, showed significant base composition heterogeneity between samples from the northern and southern waters (Fisher's exact-test, p < 0.01). Nucleotide substitutions at the three sites converged on an apical clade, which consisted of the five southern individuals, whereas its sister clade consisted only of the three northern individuals. This phylogenetic information corroborates previous ecological studies indicating the presence of separate stocks in the northern and southern waters.