Zooplankton diversity monitoring strategy for the urban coastal region using metabarcoding analysis.

Marine ecosystems in urban coastal areas are exposed to many risks due to human activity. Thus, long-term and continuous monitoring of zooplankton diversity is necessary. High-throughput DNA metabarcoding has gained recognition as an efficient and highly sensitive approach to accurately describing the species diversity of marine zooplankton assemblages. In this study, we collected 30 zooplankton samples at about 2-week intervals for 1 year. Zooplankton diversity showing a typical four season pattern. Of the "total" and "common" zooplankton, we assigned 267 and 64 taxa. The cluster structure and seasonal diversity pattern were rough when only the "common" zooplankton was used. Our study examined how to maximize the benefits of metabarcoding for monitoring zooplankton diversity in urban coastal areas. The results suggest that to take full advantage of metabarcoding when monitoring a zooplankton community, it is necessary to carefully investigate potential ecosystem threats (non-indigenous species) through sufficient curation rather than disregarding low-abundance operational taxonomic units.

DNA barcoding of brackish and marine water fishes and shellfishes of Sundarbans, the world's largest mangrove ecosystem.

The present study aims to apply a DNA barcoding tool through amplifying two mitochondrial candidate genes i.e., COI and 16S rRNA for accurate identification of fish, aquatic molluscs and crustaceans of Sundarbans mangrove wetland, to build a reference library of fish and shellfishes of this unique ecosystems. A total of 185 mitochondrial COI barcode sequences and 59 partial sequences of the 16S rRNA gene were obtained from 120 genera, 65 families and 21 orders of fish, crustaceans and molluscs. The collected samples were first identified by examining morphometric characteristics and then assessed by DNA barcoding. The COI and 16S rRNA sequences of fishes and crustaceans were clearly discriminated among genera in their phylogenies. The average Kimura two-parameter (K2P) distances of COI barcode sequences within species, genera, and families of fishes are 1.57±0.06%, 15.16±0.23%, and 17.79±0.02%, respectively, and for 16S rRNA sequences, these values are 1.74±.8%, 0.97±.8%, and 4.29±1.3%, respectively. The minimum and maximum K2P distance based divergences in COI sequences of fishes are 0.19% and 36.27%, respectively. In crustaceans, the K2P distances within genera, families, and orders are 1.4±0.03%, 17.73±0.15%, and 22.81±0.02%, respectively and the minimum and maximum divergences are 0.2% and 33.93%, respectively. Additionally, the present study resolves the misidentification of the mud crab species of the Sundarbans as Scylla olivacea which was previously stated as Scylla serrata. In case of molluscs, values of interspecific divergence ranges from 17.43% to 66.3% in the barcoded species. The present study describes the development of a molecular and morphometric cross-referenced inventory of fish and shellfish of the Sundarbans. This inventory will be useful in future biodiversity studies and in forming future conservation plan.

Metabarcoding of Fish Larvae in the Merbok River Reveals Species Diversity and Distribution Along its Mangrove Environment.

The Merbok River (north-west of Peninsular Malaysia) is a mangrove estuary that provides habitat for over 100 species of fish, which are economically and ecologically important. Threats such as habitat loss and overfishing are becoming a great concern for fisheries conservation and management. The identification of larval fish in this estuarine system is important to complement information on the adults. This is because the data could inform the spawning behaviour, reproductive biology, selection of nursery grounds and migration route of fish. Such information is invaluable for fisheries and aquatic environmental monitoring, and thus for their conservation and management. However, identifying fish larvae is a challenging task based only on morphology and even traditional DNA barcoding. To address this, DNA metabarcoding was utilised to detect the diversity of fish in the Merbok River. To complete the study, the fish larvae were collected at six sampling sites of the river. The extracted larval DNA was amplified for the Cytochrome Oxidase subunit 1 (COI) and 12S ribosomal RNA (12S rRNA) genes based on the metabarcoding approach using shotgun sequencing on the next-generation sequencing (NGS) Illumina MiSeq platform. Eighty-nine species from 65 genera and 41 families were detected, with Oryzias javanicus, Oryzias dancena, Lutjanus argentimaculatus and Lutjanus malabaricus among the most common species. The lower diversity observed from previous morphological studies is suggested to be mainly due to seasonal variation over the sampling period between the two methods and limited 12S rRNA sequences in current databases. The metabarcode data and a validation Sanger sequencing step using 15 species-specific primer pairs detected three species in common: Oryzias javanicus, Decapterus maruadsi and Pennahia macrocephalus. Several discrepancies observed between the two molecular approaches could be attributed to contaminants during sampling and DNA extraction, which could mask the presence of target species, especially when DNA from the contaminants is more abundant than the target organisms. In conclusion, this rapid and cost-effective identification method using DNA metabarcoding allowed the detection of numerous fish species from bulk larval samples in the Merbok River. This method can be applied to other sites and other organisms of interest.

DNA barcoding of coral reef fishes from Chuuk State, Micronesia.

The fish diversity of Chuuk Micronesia is currently under threat due to rapid changes in the coral reef ecosystem. Thus, accurate fish identification using DNA barcodes is fundamental for exploring species biodiversity and resource protection. In this study, we analyzed 162 fish mitochondrial DNA cytochrome c oxidase I (COI) barcodes from Chuuk Micronesia. Consequently, we identified 95 species from 53 genera in 26 families and seven orders. The average Kimura 2-parameter genetic distances within species, genera, families, and orders were calculated as 0.17%, 11.78%, 15.63%, and 21.90%, respectively. Also, we have utilized DNA barcodes to perform genetic divergence and phylogenetic analysis of families recognized as dominant groups in Chuuk State. Our findings confirm that DNA barcodes using COI are an effective approach in identifying coral reef fish species. We anticipate that the results of this study will provide baseline data for the protection of coral reef fish biodiversity at Chuuk Micronesia.

Effects of Balneotherapy in Jeju Magma-Seawater on Knee Osteoarthritis Model.

Balneotherapy is a common non-pharmacological treatment for osteoarthritis (OA), however, the efficacy is controversial in knee OA. Jeju magma-seawater (JMS) has high contents of various minerals, which has anti-inflammatory and antioxidant properties via an oral route. Thus, we examined the effects of JMS bathing on knee OA and the combination effects with diclofenac sodium as an anti-inflammatory drug. Knee OA was induced by transection of the anterior cruciate ligament and the partial meniscectomy in rat. The rats were administered subcutaneously saline or diclofenac sodium in saline, followed by bathing in thermal distilled water or JMS for 8 weeks. The model represented the characteristic changes of the cartilage degradation, osteophyte formation and synovial inflammation, and the relevant symptoms of the joint swelling and stiffness. However, the JMS bathing reduced the joint thickness and improved the mobility. It also contributed to a well-preserved tissue supported by increases in bone mineral density of the joint and decreases in Mankin scores in the cartilages. The effects involved anti-inflammation, chondroprotection, anti-apoptosis, and chondrogenesis. Overall, the JMS bathing in combination with diclofenac sodium showed a similar trend associated with synergic effects. It suggests that JMS bathing can be promising for a clinical use in knee OA.

Structure, evolution and phylogenetic informativeness of eelpouts (Cottoidei: Zoarcales) mitochondrial control region sequences.

Control region (CR) is a major non-coding domain of mitochondrial DNA in vertebrates which contains the promoters for replication and transcription of mitochondrial genome along with the binding sites for metabolic machinery and, hence, is a vital element for the integrity of mitochondrial genome as a biological replicator. The origin and diversity of structural elements within CR have been intensively studied in recent years with the involvement of new diverse taxa. In this paper, we provide new data on the nucleotide and structural patterns of CR evolution and phylogenetic suitability among eelpouts (Cottoidei: Zoarcales). To achieve this, we carried out a comparative phylogenetic and structural analysis of 29 CR sequences belonging to the long shanny Stichaeus grigorjewi together with nine sequences of other eelpouts taxa representing four families in contrast to mitochondrial protein-coding fragments. The CR organization within S. grigorjewi, as well as in all other eelpouts, is consistent with the common three-domain structure known from most vertebrates. We found a hidden CR variation constrains on the landscape level and a lack of nucleotide saturation. Finally, our results demonstrate the advantage of the length variation in CR sequences for phylogenetic reconstructions among eelpouts.

Bathing effects of east saline groundwater concentrates on allergic (atopic) dermatitis-like skin lesions induced by 2,4-dinitrochlorobenzene in hairless mice.

In the present study, it was evaluated whether east saline groundwater concentration solution (ESGWc) exerted a favorable inhibitory effect on 2,4-dinitrochlorobenzene (DNCB)-induced allergic/atopic-like dermatitis (AD). AD was induced and boosted by sensitization with DNCB via topical application on the dorsal back skins. Mice with DNCB-induced AD were bathed in 100-, 200- and 400-fold diluted ESGWc. After 6 weeks bathing, changes to body weight, clinical skin severity scores, scratching behavior, serum total immunoglobulin (Ig)E levels, submandibular lymph node and spleen weights, splenic cytokine levels, skin cytokine mRNA expressions, antioxidant defense systems and superoxide anion productions were recorded to determine the effects of bathing on the histopathology of dorsal back skin tissues. All DNCB-induced mice demonstrated that the induction of AD through IgE-mediated hypersensitivities, oxidative stresses, activation of MMP and apoptosis of keratinocytes resulted in no significant differences in body weight between the different groups at each time point following initial sensitization. However, markers of DNCB-induced AD were significantly inhibited (P<0.05) in a concentration-dependent manner following bathing in all concentrations of ESGWc. The results obtained in the present study suggest that bathing in ESGWc may have favorable protective effects against DNCB-induced AD due to favorable systemic and local immunomodulatory effects, active cytoprotective anti-apoptotic effects, inhibitory effects of matrix metalloproteinase activity, and anti-inflammatory and antioxidative effects.

Genetic and Structural Characterization of a Thermo-Tolerant, Cold-Active, and Acidic Endo-ß-1,4-glucanase from Antarctic Springtail, Cryptopygus antarcticus.

The CaCel gene from Antarctic springtail Cryptopygus antarcticus codes for a cellulase belonging to the glycosyl hydrolase family 45 (GHF45). Phylogenetic, biochemical, and structural analyses revealed that the CaCel gene product (CaCel) is closely related to fungal GHF45 endo-ß-1,4-glucanases. The organization of five introns within the open reading frame of the CaCel gene indicates its endogenous origin in the genome of the species, which suggests the horizontal transfer of the gene from fungi to the springtail. CaCel exhibited optimal activity at pH 3.5, retained 80% of its activity at 0-10 °C, and maintained a half-life of 4 h at 70 °C. Based on the structural comparison between CaCel and a fungal homologue, we deduced the structural basis for the unusual characteristics of CaCel. Under acidic conditions at 50 °C, CaCel was effective to digest the green algae (Ulva pertusa), suggesting that it could be exploited for biofuel production from seaweeds.

Leukemic infiltration presenting as myocardial hypertrophy after complete remission of acute myeloid leukemia.

Here, we report a rare case of isolated leukemic infiltrate of the myocardium (extramedullary involvement) presenting as restrictive cardiomyopathy in a patient in complete remission of acute myeloid leukemia. It was evaluated with multimodality imaging studies (echocardiography and cardiac MRI) and further confirmed by pathology. The present case highlights the importance of maintaining a high degree of clinical suspicion when evaluating patients with progressive ventricular hypertrophy of unknown cause, including recognition of the potential involvement by recurrent hematologic malignancy.

Complete mitochondrial genome of the mottled skate: Raja pulchra (Rajiformes, Rajidae).

The complete sequence of mitochondrial DNA of a mottled skate, Raja pulchra was sequenced as being circular molecules of 16,907 bp including 2 rRNA, 22 tRNA, 13 protein-coding genes (PCGs), and an AT-rich control region. The organization of the PCGs is the same as those found in other Rajidae species. The nucleotide of L-strand is composed of 29.8% A, 28.0% C, 27.9% T, and 14.3% G with a bias toward A + T slightly. Twelve of 13 PCGs are initiated by the ATG codon while COX1 starts with GTG. Only ND4 harbors the incomplete termination codon, TA. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA with the exception of [Formula: see text] which has a reduced DHU arm. This mitogenome will provide essential information for better phylogenetic resolution and precision of the family Rajidae and the genus Raja as well as for establishment of a fish stock recovery plan of the species.

The F type mitochondrial genome of hard-shelled mussel: Mytilus coruscus (Mytiloida, Mytilidae).

We determined the complete mitochondrial genome of the female hard-shelled mussel Mytilus coruscus (Gould, 1869) (F type). The F type genome is composed of 16,642 bp in length including 12 protein coding genes (PCGs), 2 rRNA and 23 tRNA with the same gene content and order as the other Mytilus species which is characterized by the absence of the ATPase8 gene and addition of tRNA(Met) (AUA). The nucleotide composition of the genome shows that the percentage of A+T (63.2%) is higher than those of M. edulis complex species (M. edulis, M. galloprovincialis, M. trossulus) but lower than that of M. californianus. The F type mitochondrial genome of M. coruscus will provide useful information on the evolutionary history of the Mytilus species in the family Mitilidae.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

Complete mitochondrial genome sequence of Heliocidaris crassispina (Camarodonta, Echinometridae).

The whole mitochondrial genome sequence of sea urchin Heliocidaris crassispina of the family Echinometridae is determined for the first time in this study. The circular mitogenome (15,702 bp) consists of typical Camarodonta gene order and its components including 2 rRNA, 22 tRNA, 13 protein-coding genes and a control region. Phylogenetic analysis based on the 13 concatenated protein-coding gene sequences shows that H. crassispina is closer to the species of Strongylocentrotidae than Parechinidae, but the separation between H. crassispina and the Strongylocentrotid species occurred early in their evolution. The complete mitochondrial genome presented in this study is useful for inferring the phylogenetic relationship among the families of Echinidea sea urchins.

The complete mitochondrial genome of the marine polychaete: Hediste diadroma (Phyllodocida, Nereididae).

The mitogenome sequence of a polychaete Hediste diadroma (Phyllodocida, Nereididae) was determined first in the genus Hediste. The circular genome is 15,765bp in size including 13 protein-coding genes (PCGs), 2 rRNA and 22 tRNA of which the order and structure is same as those of other Nereididae species. All PCGs use ATG as the start codon while for the stop codon, COI, ND5 and ND1 use an incomplete codon of T. The genome consists of 32.4% A, 18.2% C, 14.2% G, 35.2% T, showing a high content of A + T similar to the other Phyllodocid polychaetes.

Bathing Effects of Various Seawaters on Allergic (Atopic) Dermatitis-Like Skin Lesions Induced by 2,4-Dinitrochlorobenzene in Hairless Mice.

We evaluated the preventive effects of four types of seawater collected in Republic of Korea on hairless mice with 2,4-dinitrochlorobenzene- (DNCB-) induced allergic/atopic dermatitis (AD). The anti-inflammatory effects were evaluated by measuring tumor necrosis factor- (TNF-) α and interleukins (ILs). Glutathione (GSH), malondialdehyde (MDA), superoxide anion, and inducible nitric oxide synthase (iNOS) were measured to evaluate the antioxidant effects. Caspase-3 and poly (ADP-ribose) polymerase (PARP) were observed to measure the antiapoptotic effects; matrix metalloproteinase- (MMP-) 9 levels were also evaluated. Mice with AD had markedly higher clinical skin severity scores and scratching behaviors; higher TNF-α and ILs (1ß, 10, 4, 5, and 13) levels; higher MDA, superoxide anion, caspase-3, PARP, and MMP-9 levels; and greater iNOS activity. However, the severity of AD was significantly decreased by bathing in seawaters, but it did not influence the dermal collagen depositions and skin tissue antioxidant defense systems. These results suggest that bathing in all four seawaters has protective effects against DNCB-induced AD through their favorable systemic and local immunomodulatory effects, active cytoprotective antiapoptotic effects, inhibitory effects of MMP activity and anti-inflammatory and antioxidative effects.

Morphological, molecular, and biochemical characterization of astaxanthin-producing green microalga Haematococcus sp. KORDI03 (Haematococcaceae, Chlorophyta) isolated from Korea.

A unicellular red microalga was isolated from environmental freshwater in Korea, and its morphological, molecular, and biochemical properties were characterized. Morphological analysis revealed that the isolate was a unicellular biflagellated green microalga that formed a non-motile, thick-walled palmelloid or red aplanospore. To determine the taxonomical position of the isolate, its 18S rRNA and rbcL genes were sequenced and phylogenetic analysis was performed. We found that the isolate was clustered together with other related Haematococcus strains showing differences in the rbcL gene. Therefore, the isolated microalga was classified into the genus Haematococcus, and finally designated Haematococcus sp. KORDI03. The microalga could be cultivated in various culture media under a broad range of pH and temperature conditions. Compositions of the microalgal cellular components were analyzed, and its protein, carbohydrate, and lipid compositions were estimated to be 21.1 ± 0.2%, 48.8 ± 1.8%, and 22.2 ± 0.9%, respectively. In addition, D-glucose and D-mannose were the dominant monosaccharides in the isolate, and its amino acids were composed mainly of aspartic acid, glutamic acid, alanine, and leucine. Moreover, several polyunsaturated fatty acids accounted for about 80% of the total fatty acids in Haematococcus sp. KORDI03, and the astaxanthin content in the red aplanospores was estimated to be 1.8% of the dry cell weight. To the best of our knowledge, this is the first report of an Haematococcus sp. isolated from Korea, which may be used for bioresource production in the microalgal industry.

The complete mitochondrial genome of the Longnose skate: Raja rhina (Rajiformes, Rajidae).

The complete sequence of mitochondrial DNA of a longnose skate, Raja rhina was determined for the first time. It is 16,910 bp in length containing 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of 30.1% A, 27.2% C, 28.5% T and 14.2% G, showing a slight A + T bias. The G is the least used base and markedly lower at the third codon position (5.4%). Twelve of the 13 protein coding genes use ATG as their start codon while the COX1 starts with GTG. As for stop codon, only ND4 shows incomplete stop codon TA. This mitogenome is the first report for a species of the genus Raja, and providing a valuable resource of genetic information for understanding the phylogenetic relationship and the evolution of the genus Raja as well as the family, Rajidae.

Complete mitochondrial genome of Chilean sea urchin: Loxechinus albus (Camarodonta, Parechinidae).

The complete mitochondrial genome of Chilean sea urchin Loxechinus albus, the single species of the genus Loxechinus, is determined. The circular mitogenome is 15,709 bp in length containing 2 rRNA, 22 tRNA and 13 protein coding genes as well as the control region. The gene order is identical to those of described Camarodonta species. There are 24 bp gene overlaps at 6 locations and 124 bp intergenic spacers at 17 boundaries. The nucleotide composition of the genome is 31.2% A, 22.3% C, 29.7% T, and 16.8% G. The A+T bias (60.9%) is similar to that of P. lividus (60.3%) but slightly higher than those of strongylocentrotid species (58.8-59.8%). The mitogenome sequence of L. albus will provide valuable information on the phylogeny and evolution of the genus Loxechinus in relation to other Camarodonta sea urchins.

The complete mitochondrial genome of the marine polychaete: Perinereis aibuhitensis (Phyllodocida, Nereididae).

We determined the complete mitochondrial genome sequence of the marine polychaete Perinereis aibuhitensis (Grube, 1878) (Phyllodocida, Nereididae), a dominant species in the mudflat of the West Pacific and Indian Ocean. The complete genome of P. aibuhitensis is 15,852 bp in size including 13 protein coding genes (PCGs), 2 rRNA and 22 tRNA with the same gene order and structure as those of other Nereididae species. The nucleotide composition is 29.5% A, 21.2% C, 14.1% G, 35.2% T, showing a high content of A+T with G being used least in the third codon position (6.7%). All PCGs use ATG as the start codon while for the stop codon COI and ND1 use incomplete codon of T. The mitogenome sequence of P. aibuhitensis is second to that of P. nuntia known in the genus Perinereis, which will provide useful information for understanding evolutionary history of the genus Perinereis within the family Nereididae.

Complete mitochondrial genome of the Kwangtung skate: Dipturus kwangtungensis (Rajiformes, Rajidae).

The complete sequence of mitochondrial DNA of a Kwangtung skate, Dipturus kwangtungensis, was determined as being circular molecules of 16,912 bp including 2 rRNA, 22 tRNA, 13 protein coding genes (PCGs) and a control region. The arrangement of the PCGs is the same as that found in other Rajidae species. The nucleotide of L-strand which encodes most of the proteins is composed of 30.2% A, 27.4% C, 28.2% T and 14.2% G with a bias toward A+T slightly. Twelve of 13 PCGs are initiated by the ATG codon while COX1 starts with GTG. Only ND4 harbors the incomplete termination codon, TA. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA with the exception of tRNA(Ser)AGY, which has a reduced DHU arm. This mitogenome is the first report for a species of the genus Dipturus, which will become an important source of information on the phylogenetic relationship and the evolution of the genus Dipturus within the family Rajidae.