Functional analysis of LFRFamide signaling in Pacific abalone, Haliotis discus hannai.

The invertebrate LFRFamide (LFRFa) and short neuropeptide F (sNPF), consisting of 6 to 10 amino acids, are orthologs for bilaterian NPF/Y, which consist of 36 to 40 amino acids. Recently, a molluscan G protein-coupled receptor (GPCR) for NPF was characterized in Pacific abalone (Haliotis discus hannai). To address the functional evolutionary route of the invertebrate LFRFa and NPF signaling system, in this study, we identified cDNAs encoding LFRFa precursors and the sNPF receptor (Hdh-sNPFR) in Pacific abalone. Four LFRFa mature peptides with 6 or 7 amino acids were predicted: GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa. Hdh-sNPFR was identified as a classical rhodopsin-like GPCR and classified into a molluscan sNPFR group. In HEK293 cells, Hdh-sNPFR was mainly localized in the cell membranes and internalized in the cytoplasm following treatment with LFRFa peptides. Reporter assays demonstrated that LFRFa peptides inhibit forskolin-stimulated cAMP accumulation in Hdh-sNPFR-expressing HEK293 cells. LFRFa precursor and Hdh-sNPFR transcripts were more strongly expressed in the cerebral and pleural-pedal ganglia of Pacific abalone than in the peripheral tissues such as the ovary, gills, intestine, and hepatopancreas. The levels of LFRFa transcripts in the ovary, intestine, and hepatopancreas were significantly higher in mature female abalone than in immature females. Injection of LFRFa induced the egg release and spawning behavior of mature abalone, but suppressed food intake. These results suggest that LFRFa peptides are endogenous ligands for Hdh-sNPFR involved in food intake and reproduction through a Gαi-protein dependent signaling pathway.

Gonadal Abnormality and Intersexuality of Oplegnathus fasciatus (Teleostei: Oplegnathidae) Collected from the Southern Coast of Korea: A Case Report.

We report on the gonadal structure abnormality and intersexuality found from a survey regarding the reproductive potential of the rock bream, Oplegnathus fasciatus, along the southern coast of Korea. In total, 448 samples were collected for histological analysis (total length 27.5±6.8 cm; total weight 522.9±385.6 g). The sex ratio (F:M) was 1:0.46 (n=307:141), with 68.5% being females. The frequency of oocyte atresia and ovarian neoplasm in the females was 85.3% and 21.5%, respectively, and the frequency of testicular lobule and cyst deformation and testicular neoplasm in the males was 73.1% and 37.6%, respectively. The scale formation in the ovary and testis was approximately 2.0% for both sexes. The intersexuality was 42.2%, with the intersexuality among females being 49.5%, which was significantly higher than that of males (26.2%). Such findings indicate a negative impact on the reproductive output of the rock bream along the southern coast of Korea; however, a more detailed study is required to ascertain accurate causes.

Toxic effects of juvenile sablefish, Anoplopoma fimbria by ammonia exposure at different water temperature.

Juvenile sablefish, Anoplopoma fimbria (mean length 17.1±2.4cm, and mean weight 75.6±5.7g) were used to evaluate toxic effects on antioxidant systems, immune responses, and stress indicators by ammonia exposure (0, 0.25, 0.75, and 1.25mg/L) at different water temperature (12 and 17°C) in 1 and 2 months. In antioxidant responses, superoxide dismutase (SOD) and catalase (CAT) were significantly increased by ammonia exposure, whereas glutathione (GSH) was decreased. In immune responses, lysozyme and phagocytosis activity were significantly increased by ammonia exposure. In stress indicators, plasma glucose, heat shock protein 70 (HSP 70), and cortisol were significantly increased. At high water temperature (17°C), alterations by ammonia exposure were more distinctly. The results of this study indicated that ammonia exposure can induce toxic effects in the sablefish, and high water temperature can affect the ammonia exposure toxicity.

Characterization and spatiotemporal expression of gonadotropin-releasing hormone in the Pacific abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in humans and other vertebrates. Recently, GnRH-like cDNAs and peptides were reported in marine mollusks, implying that GnRH-mediated reproduction is an ancient neuroendocrine system that arose prior to the divergence of protostomes and deuterostomes. Here, we evaluated the reproductive control system mediated by GnRH in the Pacific abalone Haliotis discus hannai. We cloned a prepro-GnRH cDNA (Hdh-GnRH) from the pleural-pedal ganglion (PPG) in H. discus hannai, and analyzed its spatiotemporal gene expression pattern. The open reading frame of Hdh-GnRH encodes a protein of 101 amino acids, consisting of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. This structure and sequence are highly similar to GnRH-like peptides reported for mollusks and other invertebrates. Quantitative polymerase chain reaction demonstrated that Hdh-GnRH mRNA was more strongly expressed in the ganglions (PPG and cerebral ganglion [CG]) than in other tissues (gonads, gills, intestine, hemocytes, muscle, and mantle) in both sexes. In females, the expression levels of Hdh-GnRH mRNA in the PPG and branchial ganglion (BG) were significantly higher at the ripe and partial spent stages than at the early and late active stages. In males, Hdh-GnRH mRNA levels in the BG showed a significant increase in the partial spent stage. Unexpectedly, Hdh-GnRH levels in the CG were not significantly different among the examined stages in both sexes. These results suggest that Hdh-GnRH mRNA expression profiles in the BG and possibly the PPG are tightly correlated with abalone reproductive activities.

Decalcification of benthic foraminifera due to "Hebei Spirit" oil spill, Korea.

In order to determine the effects on foraminifera due to spilled crude oil in the "Herbei Spirit" incident, a study of benthic foraminiferal assemblages was carried out on sediment samples collected from the Sogeunri tidal flat, Taean Peninsula, Korea. Breakages of the chambers in the Ammonia beccarii and Elphidium subincertum species of the Sogeunri tidal flat with a low pH (6.98 on average) were marked. These chamber breakages occurred in 71.6% of A. beccarii and are thought to be caused by decalcification due to the fall in pH resulting from the "Hebei Spirit" oil spill. The factors that affect breakage of the chamber in benthic foraminifera under low pH condition may be not only deto decalcification but also to exposure duration of substrata in the tidal flat spilled crude oil.

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae).

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.

Identification of estrogen-like effects and biologically active compounds in river water using bioassays and chemical analysis.

The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: S1; hot spot points: S2-1, S2-2, and S2-3; and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17beta-E(2)-equivalent quantities) ranging from 25.35-677.15 pg/L. Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals, 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 x1 0(-5)) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 microg/L in S2-2) that can induce VTG induction in fish (>1 microg/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.

Environmental estrogenic effects and gonadal development in wild goldfish (Carassius auratus).

Serum vitellogenin (VTG) contents of wild goldfish (Carassius auratus) were investigated as a sensitive biomarker for artificial estrogenic compounds in aquatic environments. Goldfish was sampled from a pristine area, a river situated 5 km downstream from a sewage treatment works (STW), and also from the Young-San River in Korea. The female yolk precursor protein VTG was not detected when gonadosomatic index (GSI) was less than 0.85%, while VTG levels of >10 microg/ml were found in males whose GSI was less than 1.53%. In male goldfish sampled from STW and the Young-San River, the higher VTG corresponded to lower GSI. This study suggested a trend that gonad development was connected to VTG levels in both sexes, and the application of GSI and histological analysis provide an attractive possibility that it could be included in the panel of markers used for estrogenic activity investigation of aquatic environments.