Stomach contents of the Antarctic toothfish, Dissostichus mawsoni, collected from subareas 58.4 and 88.3, were analyzed using next generation sequencing (NGS) technology. After processing the raw reads generated by the MiSeq platform, a total of 131,233 contigs (130 operational taxonomic units [OTUs]) were obtained from 163 individuals in subarea 58.4, and 75,961 contigs (105 OTUs) from 164 fish in subarea 88.3. At 98% sequence identity, species names were assigned to most OTUs in this study, indicating the quality of the DNA barcode database for the Antarctic Ocean was sufficient for molecular analysis, especially for fish species. A total of 19 species was identified from the stomach of D. mawsoni in this study, which included 14 fish species and five mollusks. More than 90% of contigs belonged to fish species, supporting the postulate that the major prey of D. mawsoni are fish. Two fish species, Macrourus whitsoni and Chionobathyscus dewitti, were the most important prey items (a finding similar to that of previous studies). We also obtained genotypes of prey items by NGS analysis, identifying an additional 17 representative haplotypes in this study. Comparison with three previous morphological studies and the NGS-based molecular identification in this study extended our knowledge regarding the prey of D. mawsoni, which previously was not possible. These results suggested that NGS-based diet studies are possible, if several current technical limitations, including the quality of the barcode database or the development of precise molecular quantification techniques to link them with morphological values, are overcome. To achieve this, additional studies should be conducted on various marine organisms.

We developed a cost-effective metabarcoding strategy to analyze phytoplankton community structure using the Illumina MiSeq system. The amplicons (404–411 bp) obtained by end-pairing of two reads were sufficiently long to distinguish algal species and provided barcode data equivalent to those generated with the Roche 454 system, but at less than 1/20th of the cost. The original universal primer sequences targeting the 23S rDNA region and the PCR strategy were both modified, and this resulted in higher numbers of eukaryotic algal sequences by excluding non-photosynthetic proteobacterial sequences supporting effectiveness of this strategy. The novel strategy was used to analyze the phytoplankton community structure of six water samples from the East/Japan Sea: surface and 50 m depths at coastal and open-sea sites, with collections in May and July 2014. In total, 345 operational taxonomic units (OTUs) were identified, which covered most of the prokaryotic and eukaryotic algal phyla, including Dinophyta, Rhodophyta, Ochrophyta, Chlorophyta, Streptophyta, Cryptophyta, Haptophyta, and Cyanophyta. This highlights the importance of plastid 23S primers, which perform better than the currently used 16S primers for phytoplankton community surveys. The findings also revealed that more efforts should be made to update 23S rDNA sequences as well as those of 16S in the databases. Analysis of algal proportions in the six samples showed that community structure differed depending on location, depth and season. Across the six samples evaluated, the numbers of OTUs in each phylum were similar but their relative proportions varied. This novel strategy would allow laboratories to analyze large numbers of samples at reasonable expense, whereas this has not been possible to date due to cost and time. In addition, we expect that this strategy will generate a large amount of novel data that could potentially change established methods and tools that are currently used in the realms of oceanography and marine ecology.

Eyestalk ablation (ESA) is commonly used in aquaculture to stimulate ovarian maturation in crustaceans, and methyl farnesoate (MF) affects crustacean molting and reproduction. To investigate the physiological effects of ESA and MF treatments on the shrimp Litopenaeus vannamei, we compared the effects of single eyestalk removal and MF injections. The ESA group had the lowest survival rate (50%), and individuals in the 0.1 µg and 1.0 µg MF-treated groups had survival rates of 80 and 73.3%, respectively. Conversely, molting numbers were highest in the ESA group, and similar to those of the 1.0-µg MF group. To investigate shrimp growth, we measured body weight during the experimental period and found that individuals in the ESA and 1.0 µg MF groups showed significant increases in body weight. Furthermore, to investigate the effects of ESA and MF treatments on gonadal maturation, the gonad somatic index (GSI) was calculated after the experiment. All treated groups (ESA and MF) had higher GSI values than the control group, but the ESA and 1.0 µg MF groups were not significantly different. Using histological ovary analysis, we determined that all treated groups showed indications of the previtellogenic stage, unlike the control group (immature stage). These results suggest that the high-MF-concentration treatment produced effects similar to those of ESA with respect to molting number, growth, and ovarian maturation.

Microbial community structures of harmful algal bloom (HAB) caused by Heterosigma akashiwo in Geoje were analyzed using the MiSeq platform. To analyze phytoplankton communities without cross-reactivity with predominant bacteria, a new phytoplankton-specific 23S universal primer set was designed by modifying two previously used ones. The new universal primer set turned out to be a useful tool for the analysis of the phytoplankton community; it showed a high specificity for phytoplankton without cross-reactivity to bacterial sequences as well as the wide taxon coverage presenting from prokaryotic cyanobacteria to eukaryotic algae. Next Generation Sequencing (NGS) data generated by two universal primer sets (16S and 23S) provided useful information about the H. akashiwo bloom. According to the 23S universal primer set, proportions of H. akashiwo increased by more than 200-fold as the bloom occurred and its numbers were high enough to detect in control sites. Its operational taxonomic units (OTUs) were detected in the bloom sites at low proportions suggesting that the 16S universal primer set may not be as effective for monitoring harmful algal blooming (HAB) as the 23S universal primer set. In addition, several abundant OTUs in Chlorophyta were not presented by the 16S universal primer set in this study. However, the 16S primer set was useful for detecting decreases in Foraminifera as HAB occurred suggesting that genomic analyses using two universal primer sets would provide more reliable data for understanding microbial community changes by various environmental or ecological events, including HAB. Genomic analyses using two universal primer sets was also useful for determining a correlation between microbial components as HAB occurred. Heterosigma akashiwo was positively correlated with other bloom species, including Karenia mikimotoi, Teleaulax amphioxeia, and bacteria in Verrucomicrobia.

We characterized the complete mitochondrial genome of Squilloides leptosquilla (Brooks, 1886) collected from the southern waters of Korea, which is newly recorded into the Korean carcinological fauna. The total mitochondrial genome length of S. leptosquilla was 16,376 bp. This circular DNA encodes 13 proteins, two ribosomal RNAs, and 22 transfer RNAs, as well as a putative control region. Compared with other decapod crustacean mitochondrial genomes, the overall A + T content was relatively high (71.1%) as those among other stomatopod species. Nine and four protein-coding genes are encoded on the H-strand and on the L-strand, respectively. The short non-coding region (210 bp) between tRNA(Glu) and tRNA(Phe) may be the good candidate as the molecular marker to discriminate S. leptosequilla from other stomatopods.