To understand the detrimental effects of triclosan on Java medaka (Oryzias javanicus) embryos, fertilized embryos were exposed to different concentrations (1, 10, 50, 100, 200, 400, 600, 800, and 1,000 μg l-1) of triclosan until hatching. Then, we examined the survival rate and developmental parameters as well as alterations in antioxidant constituents and DNA damage markers. The results showed dose-dependent mortality, hatching delays, and developmental abnormalities in the embryos. Additionally, there were significant increases in oxidative stress parameters and antioxidant responses, along with elevated DNA damage. These findings suggest that sublethal concentrations of triclosan induce toxic effects through oxidative stress on Java medaka embryos, as evidenced by changes in in vivo parameters and biochemical constituents.

Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

In this research, the marine medaka Oryzias javanicus underwent a 96 h exposure to two concentrations of the red tide dinoflagellate Karenia mikimotoi (1,000 and 5,000 cells mL-1), and the temporal variations in biochemical responses related to antioxidant and immunity parameters were assessed in the liver tissue. The study revealed a significant increase in ichthyotoxicity with elevated cell concentrations of K. mikimotoi, especially evident at 96 h in marine medaka exposed to 5,000 cells mL-1. At 1,000 cells mL-1 of K. mikimotoi, the opercular respiratory rate showed a significant increase, whereas exposure to 5,000 cells mL-1 resulted in a lowered rate. The intracellular malondialdehyde content was significantly elevated in response to both cell concentrations at 96 h. Regarding glutathione content, levels were significantly increased by exposure to both cell concentrations. Catalase and superoxide dismutase enzymatic activities experienced an increase at 1,000 cells mL-1 of K. mikimotoi, while their activities were reduced at 5,000 cells mL-1 at 96 h. The analysis of two immunity parameters, alternative complement pathway and lysozyme, demonstrated significantly reduced activities in the liver tissue exposed to 5,000 cells mL-1 of K. mikimotoi. These findings aim to enhance the understanding of K. mikimotoi toxicity in marine fish by offering insights into biochemical responses associated with harmful algal blooms.

In this study, the marine medaka Oryzias javanicus was exposed to two concentrations of non-toxin-producing red tide dinoflagellate C. polykrikoides (1,000 and 2,000 cells ml-1) for 96 h, and the time-course biochemical responses of antioxidant and immunity parameters were analyzed in the liver tissue. Significant ichthyotoxicity with increasing cell concentrations of C. polykrikoides and exposure period was observed for 96 h. Opercular respiratory rate was lowered in marine medaka exposed to 2,000 cells ml-1 of C. polykrikoides. Intracellular malondialdehyde (MDA) content significantly elevated in response to both cell concentrations. In the case of glutathione (GSH) content, the levels were significantly elevated by 1,000 cells ml-1 of C. polykrikoides, but the contents significantly depleted upon exposure to 2,000 cells ml-1 of C. polykrikoides. Similarly, enzymatic activities of catalase (CAT) and superoxide dismutase (SOD) were increased by 1,000 cells ml-1 of C. polykrikoides, whereas their activities were lowered by 2,000 cells ml-1 of C. polykrikoides. Analysis of the two immunity parameters, alternative complement pathway and lysozyme, showed significantly lowered activities in 2,000 cells ml-1 of C. polykrikoides-exposed liver tissue. These biochemical effects of C. polykrikoides on marine medaka would be helpful for understanding its acute effects in marine fish.

Larvae from the marine medaka fish Oryzias javanicus were exposed with polystyrene microplastics (MPs) for 24 h. Exposure to waterborne fluorescent MPs showed clear ingestion and egestion in feces. Under constant MPs, the concentration of dissolved oxygen significantly decreased in 24 h compared to the control. Significant intracellular reactive oxygen species and malondialdehyde contents were detected in larvae, indicating oxidative stress and lipid peroxidation. Significant elevations in mRNA expressions of heat shock protein 70 and antioxidant defense system genes (glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase) were measured with increases in enzymatic activity of oxidative stress-related proteins. Taken together, the alterations to the molecular and biochemical components suggested that waterborne MPs had an oxidative stress effect on marine medaka larvae.

The method of natural spawning is very passive and inconvenient for the study of developmental engineering in marine medaka, Oryzias dancena. The optimum concentration of human chorionic gonadotropin (HCG) and carp pituitary extract (CPE) for ovulation and spawning, and the injection time for the artificial spawning of marine medaka were analyzed in this study. The success rate, survival rate, and hatching rate were highest with 100 IU HCG kg-1 BW and 5 mg CPE L-1 in both male and female marine medaka (p<0.05). After obtaining unfertilized eggs and sperm by the injection of HCG and CPE into the broodstock of marine medaka, artificial fertilization could be successfully achieved any time fertilized eggs are needed in this species. This result should be useful for developing a study program for marine medaka as an experimental animal.

The sectioned-body morphometric characteristics of the diploid and triploid marine medaka, Oryzias dancena, of both sexes were examined to collect basic data on the significant differences between the diploid and triploid fish. Significant differences between the diploid and triploid fish in both sexes were observed in the body circumference anterior to the base of the pelvic fin, the body circumference anterior to the base of the anal fin, the body circumference anterior to the base of the dorsal fin, the area anterior to the base of the pelvic fin, the area anterior to the base of the anal fin, the area anterior to the base of the dorsal fin, the total height anterior to the base of the pelvic fin, the total height anterior to the base of the anal fin, the height anterior to the base of the pelvic fin, the height anterior to the base of the anal fin, the width anterior to the base of the anal fin, the belly thickness II anterior to the base of the anal fin, section shape 2-1, and section shape 4-1 (p<0.05). These measurements were greater in the triploid marine medaka of both sexes than those in the diploid marine medaka of both sexes, and they were also greater in the male diploid and triploid marine medaka than those in the corresponding female fish. Therefore, the sectioned-body morphometric dimensions were greater in the triploid males than those in the triploid females and the diploid fish in this study.

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

해산송사리, Oryzias dancena 2배체와 유도 3배체 각 조직에서의 세포주기를 비교, 분석하였다. G1, S, G2+M기의 분포 빈도에서 꼬리지느러미인 경우 2배체에서는 85.8%, 7.6% 및 6.9%이었으며 유도 3배체에서는 91.2%, 3.6% 및 5.2%이었다. 간 조직인 경우 2배체에서는 78.4%, 10.6%, 및 11.0%이었으며 유도 3배체는 86.2%, 5.9% 및 7.9%이었다. 아가미 조직인 경우 2배체에서는 79.3%, 9.4% 및 11.3%이었으며 유도 3배체는 85.7%, 5.4% 및 8.9%이었다. 2배체와 유도 3배체에서 조직 간 세포 주기 빈도에서 유의한 차이가 있었다 (p<0.05). Mitosis (체세포분열)은 유도 3배체에 비해 2배체가 더욱 활성이 있었으며, 이러한 체세포분열은 2배체와 유도 3배체 모두에서 꼬리지느러미 조직 보다는 간 조직과 아가미 조직에서 더욱 왕성하였다.

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including 28°C, 31°C, 34°C, and 37°C and 2 basal media including Dulbecco’s modified eagle’s medium (DMEM) and Leibovitz’s L-15 medium (L15). With the exception of a case cultured in L15 at 31°C, the rate of primary cell adherence reached 100% when the blastomeres were cultured over 31°C. The period for primary adherence was significantly shorter in the groups cultured in 34°C and 37°C than in the ones in 28°C and 31°C. The proportion of subculture was significantly high in the group cultured in DMEM at 31°C compared to the other groups. Collectively, we demonstrated that the culture in DMEM at 31°C was effective to primary culture of the blastomeres derived from blastula embryos.

제초제 alachlor에 대한 송사리 및 지렁이 독성시험을 실시하고 국내 잔류실태조사 결과를 바탕으로 생태 위해성평가를 실시하였다. 송사리 독성시험 결과, 96시간 LC50는 1.8 mg L-1이었으며, 수정란을 사용한 초기생장단계 독성시험에서는 부화율에 대한 NOEC가 100 μg L-1으로 나타났다. 한편, 지렁이 급성독성시험에서 LC50(14일)는 94.1 mg kg-1, NOEC는

송사리(Oryzias latipes)는 한국, 일본 및 중국 등을 포함하는 동북아시아에 분포하는 어종으로. 주로 담수계 독성 실험용 표준시험생물로 이용되고 있다. 본 연구는 담수에 주로 분포하는 송사리가 marine receiving water, 공극수 또는 해양투기물질과 같은 해수의 생태독성평가를 위한 표준시험생물로서 가능성을 구명하기 위하여 염분 내성 및 표준독성물질을 이용한 민감도 실험을 수행하였다. 송사리의 염분 내성을 구명하기 위하여 다양한 염

The purpose of this study is to examine fluctuating asymmetry of eye diameter, maxilla length, operculum length, and the number of pectoral fin ray and pelvic fin ray between ploidy and sex in diploid and triploid marine medaka, Oryzias dancena. In all experimental groups, eye diameter and maxilla length showed no significant difference between left side and right side (p>0.05). Results of operculum length in triploid male group and pectoral fin ray's number in diploid male group showed similarity ones with results of operculum length in triploid female group and pectoral fin ray’s number in diploid female group (p<0.05). However, the operculum length in diploid male group and pectoral fin ray's number in triploid male group showed consinderable difference with those of operculum length in diploid female group and pectoral fin in triploid female group. Findings of pelvic fin ray's number in all groups were similar to those of pectoral fin ray’s number in all groups (p<0.01).

The morphometric truss characteristics and classical dimensions of the marine medaka, Oryzias dancena, that might distinguish diploid and triploid fish were examined. Significant differences in all the classical and truss dimensions of the diploid and triploid fish were observed in both sexes (p<0.01). All the dimensions of the triploid fish were greater than those of the diploid fish. The triploid marine medaka shows sexual dimorphism in these characters, and the sexual dimorphism of the triploid marine medaka is similar to that of the diploid marine medaka. Thus, when their classical dimension and truss dimension was measured, the growth of triploid marine medaka is faster than that of the diploid fish, and it displays clear sexual dimorphism, with male fish having longer dorsal and anal fins than female fish.

Optimum concentrations of anesthetic clove oil and anesthetic lidocaine-HCl were determined for a species of adult marine medaka, Oryzias dancena, over a range of salinity conditions, and investigated in a transport simulation experiment by analyzing various water and physiological parameters. Research indicated that the higher the concentration of anesthetic at each salinity, the shorter the anesthesia time at each salinity. At each concentration, fish were anesthetized slower at water salinities over 10 ppt (P<0.05). Anesthesia time at 10 ppt was faster than any other salinity. In 10 ppt salinity, the dissolved oxygen (DO) concentrations and respiratory frequencies of the clove-oil-administered groups decreased until 48 hours (P<0.05), whereas the NH4 + and CO2 concentrations increased until 48 hours (P<0.05). In same period, the DO, NH4 +, and CO2 concentrations and respiratory frequencies all decreased as the clove oil concentration increased (P<0.05). The trends in the DO, NH4 +, and CO2 concentrations and respiratory frequencies in the lidocaine-HCl-administered groups were similar to those in the clove-oil-administered groups. In conclusion, clove oil and lidocaine-HCl are effective anesthetics, improving the transportation of the marine medaka. The results from this study will contribute to safe laboratory handling of the marine medaka, which are commonly required by many research studies and experiments.

Sexual dimorphism is the most conspicuous difference between the sexes. This study examines possible sexual dimorphism and the relative growth patterns of morphometric characteristics in the marine medaka, Oryzias dancena for their potential to help differentiate between males and females of this species. The von Bertalanffy growth parameters estimated by a non-linear regression method were L∞=30.2 mm, K=3.22/year, and τ0=-0.05. All 18 characteristics measured showed a difference between males and females from 70 days after hatching. Each of these characteristics were significantly different between sexes (ANCOVA, P<0.05), and the ratio of standard length between sexes showed that males were larger than females for all five morphometric measurements. Fin length measurements were taken for 21 distances of anal fin and 7 distances of dorsal fin between landmarks. There were all differences for all dorsal fin rays between the males and the females and there is significant difference in 70 days after their hatch when the sexual dimorphism is presented. The significant difference (P<0.05) in fin ray for male and female was more greatly seen as they grow. Male marine medaka showed more rapid growth than females, with longer length, dorsal fins and anal fins. Differences in these characteristics will be useful during experiments when it is necessary to differentiate between sexes of marine medaka.

The marine medaka, Oryzias dancena is a suitable sample as a laboratory animal because it has a small size and clearly distinguishes between female and male. Data on the growth and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are very useful for studying other species. Triploidy was induced in the marine medaka by cold shock treatment (0°C) of fertilized eggs for 45 min, applied two minutes after fertilization. The diploid and triploid male fish were larger than their female counterparts (P<0.05), and the concentrations of thyroid stimulating hormone (TSH) and thyroxine (T4) were higher in the induced triploids over 1 year (P<0.05). In both the diploid and tri-ploid groups the concentrations of TSH and T4 were higher in the male fish than in the females (P<0.05), while the testo-sterone and estradiol-17ß concentrations in the induced triploids were lower than in the diploids (P<0.05). The gonadosomatic index (GSI) of the triploid fish was lower than that for the diploids, and the GSI for females in each ploidy group were higher than that for the males. For both groups the GSI was highest at 4 months of age, and decreased thereafter to 12 months. Analysis of the gonads of one-year-old triploid fish suggested that the induction of triploidy probably causes sterility in this species; this effect was more apparent in females than in males.