척삭동물문에 속하는 붉은멍게(Halocynthia aurantium)는 우렁쉥이와 같이 유용한 양식 품종으 로 사료되지만, 발생과 생태 등 생물학적 특성에 대해 잘 알려지지 않았다. 본 연구에서는 붉 은멍게 양식을 위한 기초자료를 얻기 위해 강원도 동해 연안에 서식하는 붉은멍게의 배발생을 조사하여 근연종인 우렁쉥이와 비교하였다. 그 결과, 붉은멍게의 수정부터 난할기, 낭배기, 신 경배기의 배아 및 올챙이형 유생의 발달 단계 및 형태가 우렁쉥이와 매우 유사하였다. 붉은멍 게의 수정란은 수온 11℃에서 부화까지 약 42.1시간이 소요되어 우렁쉥이의 40.9시간과 거의 유사하였다. 부화 후 어린개체로 변태하는 데 소요되는 시간도 두 종 사이에서 매우 유사하였 다. 수온 11℃에서 부화한 두 종의 유생은 모두 약 23일이 경과해서 입수공과 출수공이 명확 하게 구분되는 어린개체로 발생하였다. 수온 변화에 따른 발생 속도는 저온에서 느렸고 고온 에서 빠른 결과를 나타냈다. 붉은멍게의 경우는 9℃에서 부화까지 평균 62.3시간, 11℃에서 42.1시간, 13℃에서 36.3시간이 소요되었다. 우렁쉥이의 경우는 평균 60.4시간, 40.9시간, 35.2시 간이 소요되었다. 붉은멍게 배아의 대부분은 수온 15℃ 이상에서 정상적으로 발생이 이루어 지지 않아 종묘생산 과정에 주의가 필요한 것으로 사료된다.

The effects of temperature and salinity on egg development and settlement of the ascidian Herdmania momus were investigated. Adult specimens were collected from the Dodu Yacht facility in Jeju Island, Korea (33°30′30.54″N, 126°27′55.46″E) in August 2018. Egg development and larval settlement were observed and recorded at 8 h intervals using a stereomicroscope, under nine temperature (10, 13, 16, 19, 22, 25, 28, 31, and 34°C), and four salinity regimens (28, 30, 32, and 34 psu). The highest hatching rate (82.8±7%) was observed at 32 psu and 25°C and the lowest hatching rate (1.0±2%) was at 34 psu and 13°C. The developmental rate (0.222±0.0994) was highest at 28 psu and 28°C, and lowest (0.016±0.008) at 30 psu and 13°C. The highest settlement success rate (77.1±5%) was at 32 psu and 25°C and the lowest (0.1±1.0%) was at 30 psu, and 13°C. The rate of settlement (0.080±0.000) was highest at 28 psu and 28°C, and lowest (0.013±0.000) at 30 psu and 13°C. Both hatching and settlement success rates increased as temperature increased and tended to decrease beyond an optimal temperature range. Herdmania momus preferred 30-34 psu salinity and 22-25°C temperature. This study provides baseline information about the life history of H. momus, and important data to control the damage caused by the increase in number and distribution of this invasive ascidian.

The extracts of AS contain in alloxanthin, halocynthiaxanthin, astaxanthin and 13 kinds of carotenoids. The aim of the study was to assess the anti-oxidant activity and cell viability of AS. The anti-oxidant activity was determined by using DPPH radical inhibition activity and superoxide dismutase (SOD)-like activity. The results of cell viability assay showed that the extracts from AS were cytotoxic at concentrations above 5.0 ㎎/㎖. This study was designed to examine inflammation induced by LPS, protection effect by UVB and the toxicity of Ascidian shell extract(ASE) as a functional cosmetic ingredient. Evaluation of embryo toxicity resulted in embryo coagulation and mortality when treated at 5.0, 10.0, 20.0 ㎎/㎖. At the lowest concentration of 1.0 ㎎/㎖, hatchability resulted in 100.0 % rate. The results of arrhythmia measurement in larvae showed similarity to the evaluation of embryo toxicity. This result demonstrated that toxicity is present at concentrations greater than 5.0 ㎎/㎖. The protective effect of ASE on LPS and UVB-induced in the zebrafish was investigated. Intracellular reactive oxygen species(ROS) generated by the exposure of zebrafish to LPS, UVB-radiation were significantly decreased after treatment with ASE at 0.1 ㎎/㎖. As a result, ASE similarly reduced UVB-induced ROS generation and cell death in live zebrafsih. Therefore, it is suggested that ASE has anti-Inflammatory effects and can possibly be used as a functional substance for skin protection in the future.

본 연구는 멍게 껍질을 100.0 % 에탄올 용매로 추출한 후 DMSO로 희석하여 색소안정성을 평가하였다. pH에 따른 흡광도와 색차계 값을 측정한 결과 pH 7.0에서 흡광도와 색도의 ±a값이 가장 안정하게 나타났다. pH 3.0에서 흡광도의 감소가 나타났고 색도의 ±a값이 감소하여 멍게 껍질 색소는 pH가 중성에 가까울수록 변색방지의 효과가 있을 것으로 판단된다. 항산화제 첨가시 색소의 흡광도가 증가하였으며 α-tocopherol과 Glutathione이 가장 우수한 효과를 나타내었다.

저염 우렁쉥이 젓갈의 풍미발현물질을 구명하기 위해 SDE apparatus, GC 및 GC/MS를 이용하여 저염 우렁쉥이 젓갈 특유의 향기성분을 추출, 분리 및 동정하였다. 5℃에서 15일간 숙성시킨 저염 우렁쉥 이 젓갈의 pH는 5.17, 염도는 8.0%, 휘발성염기질소의 함량은 23.0 mg/100 g이었다. 저염 우렁쉥이 젓갈의 향기성분으로 총 96 성분이 동정되었고, 총함량은 1,221.42 μg/100 g-cyclohexanol이었다. 이들 화합물의 종류와 함량을 group별로 분류하면 alcohol류 23종, acid류 16종, aldehyde류 15종, hydrocarbon류 29종, 방향족 화합물 6종, ester류 2종, 함질소 화합물 2종 및 기타 3종으로 구성되어 있었는데, 계수적인 측면에서 가장 많은 화합물은 hydrocarbon류였고, 양적인 측면에서 가장 많은 화합물은 1-octanol을 위주로 한 alcohol류였다. 저염 우렁쉥이 젓갈의 향기발현에는 alcohol류를 위주로 여기에 aldehyde류, acid류, 방향족 화합물, ester류 및 함질소 화합물 등이 관여하며, 이들 성분이 서로 조화를 이루어 저염 우렁쉥이 젓갈의 독특한 향기를 발현한다고 생각되었다.

In ascidians, a primitive chordate, maternal cytoplasmic factors and inductive interactions are involved in the specification of cell fate in early embryos. The larval structure of ascidians is relatively simple, and the major mesodermal tissues of the tadpole larva are notochord, muscle and mesemchyme. Formation of muscle cells is a cell-autonomous process, and localized maternal macho-1 mRNA specify muscle fate in the posterior marginal zone of the early embryo. In contrast, inductive influence from endoderm precursors plays important roles in the specification of notochord and mesenchyme fates. FGF-Ras-MAPK signaling is involved in the induction of both tissues. The difference in responsiveness of the posterior mesenchyme and anterior notochord precursors is caused by the presence or absence of the posterior-vegetal egg cytoplasm, respectively. In these cases, directed signal may polarizes the responding cells and cause asymmetric cell divisions that operate in both the anterior and posterior regions.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. It is, however, still unclear what signaling and molecular event control polarized distribution of mitochondria in the early ascidian embryonic development. To obtain molecular markers for studying mechanisms involved in polarized distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in all cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like a mesh structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. These antibodies showed that mitochondria were distributed evenly in the animal hemisphere blastomeres at cleavage stages, whereas did not in the vegetal hemisphere blastomeres. Mitochondria were transferred more into cells of the marginal zone, such as muscle and nerve cord lineage cells, than into cells of the central zone, such as mesenchyme, notochord and endoderm lineage, in the vegetal hemisphere. Therefore, it is suggested that these antibodies may be useful as markers for analysing mechanisms involved in polarized distribution of mitochondria during ascidian embryogenesis.

Asymmetric cell divisions play crucial roles during ascidian embryogenesis. In these processes, an FGF signaling is an essential inductive signal for establishing cell fate polarization, such as mesenchyme and notochord. It was well reported that the FGF signaling cascade is composed of FGF, FGF receptor, Ras, MEK, Erk and Ets. However, mechanisms of communication between the FGF and other signaling pathways and of integrated regulation of signaling pathways have remained largely unknown. In this study, we isolated HrS6K, a homologue of the S6K gene that belongs to the S6-H1 kinase of the ribosomal S6 kinase family, and HrNck1, a homologue of Nck1 gene that encodes an adaptor protein containing Src homology 2 and 3 domains, from the ascidian Halocynthia roretzi, to elucidate the mechanisms. Zygotic expression of HrS6K was initiated as early as the 16-cell stage. In the 64-cell stage embryos, expression of HrS6K was seen in mesenchyme precursor cells. The signal was detected in dorsal midline cells and mesenchyme clusters of the early tailbud embryos, and then down-regulated by the late tailbud stage. In adults, HrS6K mRNA was highly detected in muscle and stomach by QPCR method. On the other hand, HrNck1 transcripts are detected maternally. Zygotic HrNck1 mRNA was strongly expressed in mesenchyme clusters of the neurula, and in tail tip cells of the early tailbud embryos. These results suggest that HrS6K and HrNck1 are involved in formation of mesenchyme cells, which are specified by the FGF signaling.

Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers produced 401 total loci in the Styela clava (SC) species, 390 in the Halocynthia roretzi (HR) and 434 in the Styela plicata (SP), respectively. Seven oligonucleotides primers generated 275 specific loci in the SC, 341 in the HR and 364 in the SP species, respectively. The oligonucleotides primer BION-23 generated 28 unique loci to each species in the SP species. Especially, the oligonucleotides primer BION-25 produced 7 unique loci to each species, which were identifying each species in the SP species. BION-17 distinguished 21 shared loci by the three ascidian species, major and/or minor fragments of sizes, which were identical in almost all of the samples. Based on the average bandsharing values of all samples, the similarity matrix ranged from 0.519 to 0.774 in the SC species, from 0.261 to 0.683 in the HR species and from 0.346 to 0.730 in the SP species. As regards average bandsharing value (BS) results, individuals from SC species (0.661±0.081) exhibited higher bandsharing values than did individuals from HR species (0.555±0.074) (P<0.05). The dendrogram obtained by the seven oligonucleotides primers indicates three genetic groups. In three ascidian species, the shortest genetic distance (0.071) exhibiting significant molecular difference was also between individual no. 20 and no. 21 within the SP species.

In animal development, the mechanisms by which localized factors and organelles in egg cytoplasm were exactly distributed into each daughter cell are essential for formation of various cell types. During ascidian Halocynthia roretzi embryogenesis, ooplasmic mitochondria were mainly segregated into muscle and neural precursor cells. At the 32-cell stage, localized mitochondria in the B6.2 blastomeres were preferentially distributed into the B7.4 muscle precursors compared with the B7.3 mesenchyme/ notochord precursors. When the B6.2 blastomeres were isolated from the early 32-cell stage embryos and then allowed to divide 2 times of cell division, the resultant partial embryos showed symmetric distribution of mitochondria, and the partial embryos were composed of equal size cells. In normal development, cell fates of the B7.3 blastomere were correlated with the unequal cleavage of B6.2 lineage cells that normally occurs in the next two-cell division stages to produce a large B8.5 mesenchyme and a small B8.6 notochord cell. Mitochondria are distributed asymmetrically in both cells. When embryos were treated with FGF receptor inhibitor SU5402 and MEK inhibitor U0126 between the 32-cell and the early 64-cell stages, the resultant embryos showed equal cleavage pattern and symmetric distribution of mitochondria in daughter cells of the B6.2 blastomeres. However, blocking of Nodal and Notch signaling did not affect the cell division pattern and mitochondrial distribution in the B6.2 lineage blastomeres between the 32-cell and 110-cell stages. Therefore, it is likely that FGF/MEK signaling is involved in asymmetric distribution of mitochondria and unequal cleavage of the B6.2 lineage blastomeres in ascidian embryo.

폐자원인 우렁쉥이 껍질을 이용한 환경친화형 유기농 액비의 개발을 위하여 유용미생물(EM)을 이용한 우렁쉥이 껍질의 최적 발효조건과 액비의 특성을 조사한 결과 다음과 같다. EM발효 우렁쉥이 껍질 액비 제조 시 당밀의 투입량이 증가할수록 pH는 낮아지며, EC는 높아지는 것으로 나타났으며, pH는 당밀이 10% 이상 투입된 시험구에서 일정하게 유지되었다. EM발효 우렁쉥이 액비의 총 질소 함량은 발효가 경과할수록 현저히 증가하였으며, 특히 당밀이 15% 이상 투입된 시험구에서 약 220% 증가율을 나타내었다. 인의 함량은 발효 21일차에서 최고치를 나타내었으며, 칼륨의 함량은 발효가 경과할수록 증가하였다. 칼슘, 마그네슘, 나트륨의 함량 역시 발효가 경과할수록 증가하였고 당밀이 15% 이상 투입된 시험구에서 증가율이 우수한 것으로 나타났으며, 칼슘의 함량이 상대적으로 높은 것으로 나타났다. 유해성분(As, Cd, Cu, Cr, Hg, Ni, pb 및 Zn)의 함량은 시험구 모두에서 비료공정규격 기준치에 적합하였다. 우렁쉥이 껍질 액비의 유리아미노산은 총 29종이 검출되었으며 총량은 7,080.94 mg/L를 나타내었고 알라닌이 656.32 mg/L로 가장 높은 함량을 나타내었다. 이상의 결과를 미루어 볼 때 우렁쉥이 껍질을 이용한 액비화에는 당밀이 15% 이상 투입된 조건이 최적인 것으로 판단된다.

FGF9/16/20 signaling pathway specify the developmental fates of notochord, mesenchyme, and neural cells in ascidian embryos. Although a conserved Ras/MEK/Erk/Ets pathway is known to be involved in this signaling, the detailed mechanisms of regulation of FGF signaling pathway have remained largely elusive. In this study, we have isolated Hr-Erf, an ascidian orthologue of vertebrate Erf, to elucidate interactions of transcription factors involved in FGF signaling of the ascidian embryo. The Hr-Erf cDNA encompassed 3110 nucleotides including sequence encoded a predicted polypeptide of 760 amino acids. The polypeptide had the Ets DNA-binding domain in its N-terminal region. In adult animals, Hr-Erf mRNA was predominantly detected in muscle, and at lower levels in ganglion, gills, gonad, hepatopancreas, and stomach by quantitative real-time PCR (QPCR) method. During embryogenesis, Hr-Erf mRNA was detected from eggs to early developmental stage embryos, whereas the transcript levels were decreased after neurula stage. Similar to the QPCR results, maternal transcripts of Hr-Erf was detected in the fertilized eggs by whole-mount in situ hybridization. Maternal mRNA of Hr-Erf was gradually lost from the neurula stage. Zygotic expression of Hr-Erf started in most blastomeres at the 8-cell stage. At gastrula stage, Hr-Erf was specifically expressed in the precursor cells of brain and mesenchyme. When MEK inhibitor was treated, embryos resulted in loss of Hr-Erf expression in mesenchyme cells, and in excess of Hr-Erf in a-line neural cells. These results suggest that zygotic Hr-Erf products are involved in specification of mesenchyme and neural cells.

The mechanisms by which embryo exactly distributes mitochondria into the blastomeres during embryogenesis are one of the important issues in developmental biology. Although the mechanisms has been thought to be important for the proper embryonic development, our understanding has remained limited. In the present study, the distribution of mitochondria was examined in embryos of the ascidian, Halocynthia roretzi, by immunohistochemical staining with three-types of the mitochondria-specific antibodies and vital staining of mitochondria with a fluorescent probe, DiOC2(3). Results of the immunohistochemical staining coincided with that of vital staining, which is able to detect the distribution of mitochondria in cytoplasm of the embryo. Mitochondria was mainly segregated into the B4.1 posterior-vegetal blastomeres at the 8-cell stage. During the next stages, mitochondria was preferentially partitioned into cells of the B-line muscle and the A-line nerve cord precursor compared with each sister cell, endoderm in the 5th cleavage stage, and mesenchyme and notochord in the 6th cleavage stage. However, the mitochondria-rich cytoplasm is divided equally among the blastomeres of the animal hemisphere between the 8-cell and the 64-cell stages. When B6.2 blastomeres were isolated at the early 32-cell stage embryo and cultured in seawater, until control embryos reached the 64-cell stage, pattern of mitochondria distribution was similar to results of the coisolated B7.3 and B7.4 blastomeres from the 64-cell stage embryos. Therefore, it is likely that mitochondria are asymmetrically segregated into the marginal cells in the vegetal hemisphere of the ascidian embryo without cell-cell interaction.

Notch signaling plays fundamental roles in various animal development. It has been suggested that Hr-Notch, a Notch homologue in the ascidian Halocynthia roretzi, is involved in the formation of peripheral neurons by suppressing the neural fates and promoting the epidermal differentiation. However, roles of Notch signaling remain controversial in the formation of nervous system in ascidian embryos. To precisely investigate functions of Notch signaling, we have isolated and characterized Hr-Numb, a Numb homologue which is a negative regulator of Notch signaling, in H. roretzi. Maternal expression of Hr-Numb mRNAs was detected in egg cytoplasm and the transcripts were inherited by the animal blastomeres. Its zygotic expression became evident by the early neurula stage and the transcripts were detected in dorsal neural precursor cells. Suppression of Hr-Numb function by an antisense morpholino oligonucleotide resulted in larvae with defect in brain vesicle and palps formation. Similar results have been obtained by overexpression of the constitutively activated Hr-Notch forms. Therefore, these results suggest that Hr-Numb is involved in Notch signaling during ascidian embryogenesis.