The complete mitochondrial genome analysis and phylogenetic position of the short barbeled grunter Hapalogenys nigripinnis (Lobotiformes: Hapalogenyidae) from Jeju island, Korea.

This study analyzed the complete mitochondrial genome of the short barbeled grunter Hapalogenys nigripinnis (Accession number: MT374064). The complete mitogenome was 16,476 bp long and included 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. Nucleotide composition of the genome was A: 28.70%, T:27.46%, G: 15.73%, and C: 28.11%. All genes were encoded on the H-strand, except for the NADH dehydrogenase subunit (ND6) and 8 tRNA genes. When compared this sequence with the mitogenome of Chinese black grunt, Korean short barbeled grunter showed difference of 64 bp of nucleotide sequence in 20 genes. Phylogenetic tree was constructed using the neighbor-joining (NJ) method and showed the phylogenetic position of the short barbeled grunter in Korea.

The complete mitochondrial genome sequence and phylogenetic analysis for Inimicus japonicus (Scorpaeniformes: Synanceiidae).

The complete mitochondrial genome (mitogenome) of the Inimicus japonicus was analyzed by next-generation sequencing in this study. The mitogenome is 16,978 base pairs (bp) long and codes for 22 transfer RNA (tRNA) genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), and 1 non-coding control region. The overall nucleotide composition of the mitogenome is: 29.61% for A, 29.16% for T, 25.26% for C, and 15.97% for G. Twenty-two tRNAs range from 67 to 74 bp in length, and 2 rRNA (12S and 16S) were 953 and 1,687 bp long, respectively. Phylogenetic analysis by neighbor-joining (NJ) method indicated that I. japonicus showed considerable genetic similarity (82%), and had a closer relationship in the phylogenetic tree to Synanceia verrucosa.

Digestive Physiological Characteristics of the Gobiidae: - Characteristics of CCK-producing Cells and Mucus-secreting Goblet Cells of Stomach Fish and Stomachless Fish.

In this study, we investigated the characteristics of CCK-producing cells and mucus-secreting goblet cells with respect to stomach fish and stomachless fish of the Gobiidae in order to provide a basis for understanding the digestive physiology. Hairychin goby (Sagamia geneionema), which is stomachless fish, the numbers of mucus-secreting goblet cells is highest in the posterior intestine portion (P<0.05), while CCK-producing cells are scattered throughout the intestine. Gluttonous goby (Chasmichthys gulosus), which is stomach fish, mucus-secreting goblet cells are most abundant in the mid intestine portion (P<0.05), whereas CCK-producing cells are observed only in the anterior and mid intestine portion. Trident goby (Tridentiger obscurus) which is stomach fish, mucus-secreting goblet cells were most abundant in the mid intestine portion (P<0.05). CCK-producing cells are found in the anterior and mid intestine portion. Giurine goby, Rhinogobius giurinus which is also stomach fish, the largest number of mucus-secreting goblet cells showed in anterior intestine portion except for esophagus (P<0.05). CCK-producing cells are present only in the anterior and mid intestine portion. In S. geneionema, digestive action occurs in the posterior intestine portion to protect and functions to activate digestion. In contrast, in C. gulosus, T. obscurus and R. giurinus, their digestive action occurs in the anterior and mid intestine portion to protect and functions to activate digestion. Further studies of the modes of food ingestion by these fish, the contents of their digestive tracts, and the staining characteristics of the goblet cells need to be carried out.

Early Osteological Development of Larvae and Juveniles in Red Spotted Grouper, Epinephelus akaara (Pisces: Serranidae).

We observed the osteological development of larval and juvenile red spotted grouper (Epinephelus akaara) in order to generate data for the assessment of skeletal deformities and to inform phylogenetic systematics research. Larvae and juveniles were obtained from a aquafarm in Muan-gun, Jeolla-namdo Province, Korea. The average water temperature at the time of breeding was 23.0°C and average water salinity was 33.0 psu. Freshly hatched fish larvae had not undergone any ossification, but ossification of the parasphenoid bone, which forms the base of the cranium, occurred as the juveniles reached an average body length (BL) of 2.49 mm. At the same time, ossification of the preopercle and opercle occurred in the operculum, and ossification of the maxilla, which forms the upper jaw, and the dentary bones, which form the lower jaw, began. In addition, ossification of the vertebra occurred by formation of 7 vertebral centra and the neural spine in the abdominal vertebra. When the juveniles reached an average (BL) of 5.22 mm, ossification of the nasal, lateral ethmoid, and alisphenoid bones occurred in the cranium; ossification of the endopterygoid and metapterygoid bones began in the palatine region; and ossification of the hypohyal and interhyal bones occurred in the hyoid arch. At an average (BL) of 20.9 mm, ossification of the basisphenoid bone in the cranium and the suborbital bone in the orbital region occurred. Ossification of the vertebra then occurred by the formation of long pairs of ribs from the third to the ninth abdominal vertebrae, completing osteological development.

Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary.

Monoclonal antibodies were generated against recombinant follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica; rec-FSH was produced in Escherichia coli and purified using Ni-NTA Sepharose column chromatography. In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary.

Molecular cloning and expression analysis of a new lily-type lectin in the rock bream, Oplegnathus fasciatus.

A new lily-type lectin RbLTL was identified from rock bream (Oplegnathus fasciatus) and its expression analysed. In this study, a new lily-type lectin gene (RbLTL) was cloned from rock bream using expressed sequence tag (EST) analysis. The full-length RbLTL cDNA was encoding a 117-amino acid protein. The deduced amino acid sequence of RbLTL contained all of the conserved features crucial for its fundamental structure, including B-lectin domain and three d-mannose binding sites. RbLTL mRNA was predominately expressed in the gills, with reduced expression noted in intestine tissue. Expression analysis of time series sampled fertilized eggs revealed that expression gradually increased 1, 3, 12, and 24 h: However, expression decreased at 36 h. RbLTL expression was differentially up-regulated in rock bream gills challenged with Streptococcus iniae, Edwardsiella tarda and RSIV. Our results revealed that novel rock bream lily-type lectin may be an important molecule involved in pattern recognition and pathogen elimination in the innate immunity of rock bream.

Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing.

Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.

Studies on antimicrobial activity of Poncirus trifoliata ethyl extract fraction against methicillin-resistant Staphylococcus aureus and to elucidate its antibacterial mechanism.

Traditional medicinal plants contain a wide variety of chemicals that have potent antibacterial activity. To find an alternative agent of overcoming the problems of methicillin-resistant Staphylococcus aureus (MRSA), the antibacterial mechanism of Ponciruss trifoliata against MRSA was investigated. Ethyl acetate (EtOAc)-soluble extract of P. trifoliata methanolic extract was evaluated for antibacterial activity using minimum inhibitory concentration (MIC). An EtOAc sub-fraction 08 (EA08) from silica-gel open column chromatography exhibited strong anti-MRSA activity. Apart from the study to isolate single compound from EA08, a synergistic antibacterial effect between the sub-fraction and ß-lactam antibiotics against MRSA was determined. In order to elucidate the antibacterial restoring mechanism of EA08 on MRSA, mRNA expression of mecA gene and production penicillin-binding protein 2a (PBP2a) encoded by mecA gene were monitored. EA 08 showed the strongest antibacterial activity with MIC value of 256 µg ml(-1). MIC of oxacillin against MRSA was dramatically reduced from 512 to 16 µg ml(-1) in combination with 256 µg ml(-1) of EA08. The fractional inhibitory concentration index of oxacillin was measured at 0.53 in combination with EA08 against MRSA, suggesting that EA08-oxacillin combinations exert synergetic effect against MRSA. The analysis of RT-PCR and Western blotting profiles revealed that EA08 inhibited mRNA expression of mecA gene and production PBP2a, which is a key determinant for ß-lactam antibiotic resistance, in a dose-dependent manner. These results indicated that EA08 eventually led to the reduction or inhibition of PBP2a production through translational inhibition in MRSA.

Synergistic Antibacterial Effect and Antibacterial Action Mode of Chitosan-Ferulic Acid Conjugate against Methicillin-Resistant Staphylococcus aureus.

We evaluated the synergistic antibacterial effect in combination with the chitosan-ferulic acid conjugate (CFA) and ß-lactam antibiotics, such as ampicillin, penicillin, and oxacillin, against methicillin-resistant Staphylococcus aureus (MRSA) using fractional inhibitory concentration (FIC) indices. CFA clearly reversed the antibacterial activity of ampicillin, penicillin, and oxacillin against MRSA in the combination mode. Among these antibiotics, the combination of oxacillin-CFA resulted in a ∑FICmin range of 0.250 and ∑FICmax of 0.563, suggesting that the oxacillin-CFA combination resulted in an antibacterial synergy effect against MRSA. In addition, we determined that CFA inhibited the mRNA expression of gene mecA and the production of PBP2a, which is a key determinant for ß-lactam antibiotic resistance, in a dosedependent manner. Thus, the results obtained in this study supported the idea on the antibacterial action mechanism that oxacillin will restore the antibacterial activity against MRSA through the suppression of PBP2a production by CFA.

Stress responses of starry flounder, Platichthys stellatus (Pallas) following water temperature rise.

Stress responses of starry flounder, Platichthys stellatus (Pallas) following water temperature rise were investigated to establish the influence of ambient temperature on this species. The physiological indicators of stress were plasma cortisol, glucose, aspartate aminotransferase, alanine aminotransferase, sodium, chloride, osmolality and triiodothyronine (T3). No significant difference in plasma parameters were observed among the experimental groups of 15 degrees C, 18 degrees C and 21 degrees C. Level of plasma cortisol (49.0-95.0 ng ml(-1)) and glucose (56.1-58.1 mg dl(-1)) of starry flounders kept at 24 degrees C-27 degrees C were significantly higher than those (cortisol: 20.4-23.6 ng ml(-1), glucose: 40.6-47.1 mg dl(-1)) observed in the 15 degrees C-21 degrees C groups. Changes in aspartate aminotransferase and alanine aminotransferase following water temperature rise showed a similar pattern to plasma cortisol and glucose. Starry flounders exposed to 27 degrees C exhibited higher plasma sodium (164.7 mmol l(-1)), chloride (147.6 mmol l(-1)), and osmolality (450.7 mOsm kg(-1)) than those (sodium: 154.0-158.7 mmol l(-1), chloride: 139.1-140.4 mmol l(-1), osmolality: 375.1-383.8 mOsm kg(-1)) fish exposed to 15-21 degrees C. Though plasma T3 (29.4 ng ml(-1)) of starry flounder increased at 24 degrees C, this hormone was significantly lower (19.3 ng ml(-1)) in fish kept at 27 degrees C than those (24.6 ng ml(-1)) the fish at 15 degrees C. This phenomenon seems to be directly associated with long-term fasting. Accordingly, the results suggested that starry flounders got stressed with osmoregulatory disturbances above 24 degrees C.

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis.

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy-Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.

Early Life History of the Sevenband Grouper, Epinephelus septemfasciatus from Korea.

This study is conducted to monitor the morphological developmental features of the egg development, larvae and juvenile of Epinephelus septemfasciatus, the fertilized eggs were gotton using artificial insemination. Matured parents are collected from marine caged fish farms in Geomun-ri, Samsan-myeon, Yeosu-si, Jeollanamdo Korea in June 2012. The fertilized eggs were pelagic eggs containing one oil globule, and measured 0.81~0.89 mm (0.85±0.04 mm, n=50) in diameter. In regard to rearing environment, the water temperature is 21.0~23.0°C and the salinity is 32.0~33.2‰. Hatching was observed from 48 hours after fertilization, the mouth and anus of prelarvae was not opened but had egg yolk at newly hatched. 4 days after hatching, the mouth and anus of postlarvae was opened and began to eat Rotifer and was measured 2.40~2.49 mm (2.45±0.03 mm n=10) in total length. 12 days after hatching, postlarvae was measured 3.77~4.67 mm (4.27±0.33 mm) in total length, its the second pole tide of dorsal fin and the first pole tide of pelvic fin was extended longitudinally. 71 days after hatching, juvenile was measured 40.5~45.4 mm (42.6±2.04 mm) in total length. Seven bands were observed in body, and pole tides of dorsal and pelvic fins were shortened.

Morphological Analysis of Blind-Side Hypermelanosis of the Starry Flounder, Platichthys stellatus during Early Development.

In Pleuronectiformes, blind-side malpigmentation (hypermelanosis) is common in cultured flatfishes, and is economically important. To understand the mechanism of blind-side hypermelanosis in flatfishes, we examined when the malpigmentation initially occurred, and studied how the symptoms proceeded during early development of the starry flounder, Platichthys stellatus. To assess quantitative pattern changes of blind-side skin, we observed morphological development of the whole body from 22 (total length [TL] 10.0±0.2 mm and body weight [BW] 8.8±0.57 mg) to 110 days (TL 23.4±0.7 mm, BW 193.6±23.3 mg) after hatching (DAH), and also examined the malpigmented area rate of blind-side skin and the malpigmented fish ratios. The experimental animals were reared in fiberglass-reinforced plastic (FRP) tanks in water at a temperature of 18.9±1.9°C and salinity of 32.6±0.6 psu and were fed with rotifer and Artemia nauplii from 22 to 48 DAH, and with A. nauplii and commercial feed from 49 to 110 DAH. As results, the first staining patch seen by the naked eye was observed around the area between the anus and pelvic fin or caudal edge of the trunk at 80 DAH (TL 20.6±0.5 mm, BW 112.5±8.8 mg). The pigmented area and the pigmented fish ratios were significantly increased from 80 to 110 DAH. These results indicated that malpigmentation on the blind side of starry flounder was initially observed at about 2 cm in length and 100 mg in weight, and the pigmented domain on the blind-side skin was continually broadened by the differentiation of pigmented cells (melanophores and xanthophores) with growth.

Genetic characterization of five hatchery populations of the Pacific Abalone (Haliotis discus hannai) using microsatellite markers.

The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient F(IS) ranged from 0.054 to 0.184 (mean F(IS) = 0.121 ± 0.056). The genetic differentiation across all populations was low but significant (overall F(ST) = 0.009, P < 0.01). Pairwise multilocus F(ST) tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry.

Wild and hatchery populations of Korean starry flounder (Platichthys stellatus) compared using microsatellite DNA markers.

Starry flounder (Platichthys stellatus) is an important sport and food fish found around the margins of the North Pacific. Aquaculture production of this species in Korea has increased because of its commercial value. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of starry flounder populations. In this study, 12 polymorphic microsatellite DNA markers were identified from a partial genomic starry flounder DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery starry flounder populations in Korea. All loci were readily amplified and demonstrated high allelic diversity, with the number of alleles ranging from 6 to 18 in the wild population and from 2 to 12 in the farmed population. A total of 136 alleles were detected at the 12 microsatellite loci in the two populations. The mean observed and expected heterozygosities were 0.62 and 0.68, respectively, in the hatchery samples and 0.67 and 0.75, respectively, in the wild samples. These results indicate lower genetic variability in the hatchery population as compared to the wild population. Significant shifts in allelic frequencies were detected at eight loci, which resulted in a small but significant genetic differences between the wild and hatchery populations (F(ST) = 0.043, P < 0.05). Further studies with additional starry flounder sample collections are needed for comprehensive determinations of the genetic varieties between the wild and hatchery populations. These microsatellite loci may be valuable for future population genetic studies, monitoring the genetic variation for successful aquaculture management and the preservation of aquatic biodiversity.