Chronic exposure to environmental concentrations of harmful algal bloom-forming dinoflagellates induces oxidative stress and reduces immune and hepatic functions in red seabream.

Harmful algal blooms (HABs) caused by dinoflagellates can be detrimental to aquaculture and fisheries. However, little is known regarding their ichthyotoxic effects on fish, particularly after chronic exposure to sublethal levels. In this study, significant modulations in physiology, immunity, antioxidant components, and hepatic indicators owing to non-toxin-producing dinoflagellate strains (Alexandrium affine and Cochlodinium polykrikoides) were analyzed in juvenile red seabream, Pagrus major, exposed to sublethal concentrations (0, 1, and 100 cells mL-1) for 60 days. At 60 days, higher mortality was induced by A. affine than by C. polykrikoides. Significant increases in respiration rate and plasma cortisol were observed in red seabream exposed to 100 cells mL-1 of the two dinoflagellates. Intracellular reactive oxygen species and malondialdehyde levels were significantly elevated in the gill and liver tissues in response to 100 cells mL-1 of either dinoflagellate. Immunity parameters such as alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased during exposure to 100 cells mL-1 of the two dinoflagellates. Although no significant change was observed in the gonadosomatic index, the hepatosomatic index was significantly decreased by exposure to 100 cells mL-1 of the two dinoflagellates on day 60. The significant decrease in enzymatic activities of ethoxyresorufin-O-deethylase, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase upon exposure to 100 cells mL-1 of either dinoflagellate suggested impaired hepatic function through prolonged exposure. Our results suggest that consistent exposure to sublethal concentrations of HAB-forming dinoflagellates is detrimental to fish physiology and biochemical defenses.

Changing Salinity Affects Hematological and Histological Response in Hybrids and Hybrid Triploids between River Puffer, Takifugu obscurus and Tiger Puffer, T. rubripes.

We investigated the physiological responses of the river puffer, Takifugu obscurus, the tiger puffer, T. rubripes, a hybrids of the two (river puffer×tiger puffer) and a hybrid triploids to acute changes of salinity from 30 psu to 0 psu and from 0 psu to 30 psu. The blood and plasma factors of each species were elevated for 48, 72, or 96 hrs, and thereafter decreased due to hyper-osmoregulation and hypo-osmoregulation. In hyper-osmoregulation and hypo-osmoregulation, the cortisol concentration of river puffer, hybrids, hybrid triploids and tiger puffer increased for 12 or 48 hrs, and decreased thereafter. Chloride cells in the gill filaments of each species increased with increasing salinity, and melano-macrophages in the kidney tissue of each species increased with decreasing salinity. In conclusion, the hematological and stress responses of the hybrids were between those of the river puffer and tiger puffer, and the hematological responses of the hybrid triploids were higher than those of the other groups. The stress response of the hybrids was more sensitive than that of the hybrid triploids. In all groups, the histological responses of kidney in hyper-osmoregulation were more sensitive than those in hypo-osmoregulation.

Karyotypic Analysis of Chicken Grunt, Parapristipoma trilimeatum, Small Yellow Croaker, Larimichthys polyactis, and Brown Croaker, Miichthys miiuy.

To clarify the cytogenetic features of the chicken grunt, Parapristipoma trilineatum, small yellow croaker, Larimichthys polyactis, and brown croaker, Miichthys miiuy, in Jeju, Korea, karyotype analysis was performed. The karyotype of chicken grunt and brown croaker consists of 24 pairs of telocentrics [2n=48, fundamental number (FN)=48], and small yellow croaker consists of 3 pairs of submetacentrics and 21 pairs of telocentrics (2n=48, FN=54). No evidence of polymorphism, including aneuploidy or sex-related heteromorphic chromosomes, was found in any specimens examined in this study.

Comparative Analysis of Fluctuating Asymmetry between Ploidy and Sex in Marine Medaka, Oryzias dancena.

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).

Pysiological Responses of Diploid and Triploid Far Eastern Catfish, Silurus asotus to Water Temperature Stress.

The aim of study is to contribute to this knowledge-base by investigating the respiratory function, the metabolic rate and the difference of physiological responses un-der low water temperature (20℃→15℃) stress be-tween diploid and triploid far eastern catfish, Silurus asotus. During the 48 hrs of water temperature stress exposure time, the respiratory frequencies, CO2 and NH4+ concentrations of diploid had higher values than those of triploid (p<0.05). However, pH of triploid was higher than those of diploid (p<0.05), and oxygen consumption rate was not different between diploid and triploid (p>0.05). The level of plasma cortisol and plasma glucose of triploid was lower than those of the diploid (p<0.05). However, in case of lactic acid, there were not significant between triploid and diploid (p>0.05). These results suggest that diploid was more sensitive for low water temperature stress response than triploid in this species.

Morphometric Characteristics of Diploid and Triploid Marine Medaka, Oryzias dancena.

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.

Temperature Dependent of Mitotic Interval for Grass Puffer, Takifugu niphobles.

The objective of this study was to determine the mitotic intervals (τ0) of two consecutive cell divisions and synchronous embryonic cleavage in grass puffer, Takifugu niphobles at different water temperatures (18, 20, 22, and 24℃). The color of the fertilized egg was light yellowish. The egg type was demersal and unadhesive. Egg weight was 0.09±0.002 mg. The sizes of unfertilized eggs were smaller than fertilized eggs in major axis and minor axis at 20℃ (p<0.05). The size of the fertilized egg of 18℃ water temperature group at the blastodisc stage was the smallest (p<0.05), but no significant differences were observed in the other water temperatures group except 18℃ water temperature group (p>0.05). The first cleavage stages at 18, 20, 22, and 24℃ were at 75, 90, 105, and 120 mins, respectively. As water temperature was increased, embryonic development and formation time of the first cleavage furrow were accelerated. There were negative correlation between τ0 and water temperature for grass puffer (Y=-1.225X+70.05, R2=0.988, n=10, where Y was τ0 and X was temperature). This study confirmed that successful hatching of grass puffer was related to water temperature. Chromosome manipulation will be helpful for this species using cleavage frequency and τ0.

The Effect of Sodium Nitrite Exposure on Physiological Response of Starved Far Eastern Catfish, Silurus asotus.

The experiment was conducted for 210 days to determine the effect of feeding, and starvation, and exposure to sodium nitrite (NaNO2) on the survival, physiological changes, hematological parameter, and stress response of Far Eastern catfish, Silurus asotus. The survival of the starved group was lower than that of the fed group during the experiment. Starvation resulted in retardation of growth, which provides an example of fish that failed to continue to grow and remain in a good condition. Blood analyses (cortisol and glucose) showed significant differences of stress response between the fed and starved groups exposed to NaNO2 at the conclusion of the experiment (p<0.05). These results suggest that all nutritional parameters used for starvation and feeding with NaNO2 stress in this experiment appear to be a useful index of nutritional status in Far Eastern catfish.

Starvation-induced Physiological Responses and RNA/DNA Ratios in Rock Bream, Oplegnathus fasciatus, and Olive Flounder, Paralichthys olivaceus.

In a 12-week experiment, the rock bream, Oplegnathus fasciatus, and olive flounder, Paralichthys olivaceus, were investigated to determine the effects of starvation on their physiological parameters. The protein and DNA contents of the starved fish were significantly higher than the initial values and those of the fed fish. The RNA contents and RNA/DNA ratios of the fed fish were significantly higher than those of the other groups (P<0.05). The hematocrit, hemoglobin, red blood cells (RBC), and mean corpuscular volume (MCV) of the fed rock bream were significantly higher than at baseline (P<0.05), whereas the mean corpuscular hemoglobin concentration (MCHC) of the fed fish was lower than at baseline (P<0.05). The hematocrit, hemoglobin, RBC, and MCHC of the starved group were significantly lower than the baseline values, whereas the MCV of the starved group was significantly higher than the baseline value (P<0.05). No significant difference in alanine aminotransferase was observed between the fed fish and baseline, whereas the starved fish value was significantly higher than the baseline value (P<0.05). There were no significant differences in cortisol levels. However, the glucose level in the fed group was significantly higher than the baseline level and that in the starved group was significantly lower than the baseline level (P<0.05).

Change of Various Characteristics between Spawning and Non-spawning Season in Diploid and Induced Triploid Far Eastern Catfish, Silurus asotus.

The purpose of this study is to investigate the differences fatty acids and hormonal parameters in the spawning and non-spawning season between the diploid and induced triploid Far Eastern catfish, Silurus asotus. The measured triploids were produced by cold shock for 50 min at 4℃ in May 2014, the spawning season of diploid was in May, and the non-spawning season was designated in January. Estradiol and testosterone and gonadosomatic index of diploid were higher than those of induced triploid in spawning season (P<0.05), and those of diploid in spawning season were higher than non-spawning season. On the other hand, thyroid stimulating hormone and thyroxine of induced triploid were higher than those of diploid in spawning season (P<0.05). Erythrocyte count of diploid was higher than that of induced triploid in spawning season and non-spawning seasons. Mean corpuscular volume and mean corpuscular hemoglobin of induced triploid were higher than those of diploid in both seasons (P<0.05). Percentages of total saturated fatty acids and n-3 polyunsaturated fatty acids of induced triploid were higher than those of diploid in spawning season, but those of diploid were higher in non-spawning season (P<0.05). Percentages of total mono unsaturated fatty acids and total n-6 polyunsaturated fatty acids of diploid were higher than those of induced triploids in spawning season, while those of induced triploid in non-spawning season were higher (P<0.05). Therefore, induced triploids in the spawning season tend to concentrate on growth and lipid-synthesization, whereas, diploids concentrate on reproduction and gonadal maturation rather than on growth. In non-spawning season, growth and lipid-synthesization were not significantly different between diploid and induced triploid.

Various Characteristics of Hybrid between River Puffer, Takifugu obscurus and Tiger Puffer, T. rubripes, and Their Hybrid Triploid.

A comparison of the growth, hematological values, fatty acids, and gonadal and growth hormonal changes of river puffer, Takifugu obscurus, tiger puffer, T. rubripes, their hybrids (river puffer × tiger puffer) and hybrid triploids was performed during 3 months of their early growth period. Several features were observed during these 3 months: hybrids showed the highest levels of specific growth rate, 1.48%; hybrid triploids showed the smallest change in viscera fat (P<0.05), but GSI was not significantly different among groups (P>0.05). Considering hematological parameters, hybrid triploids had increased mean corpuscular volume and mean corpuscular hemoglobin (P<0.05), but other parameters were not significantly different between groups (P>0.05). With respect to fatty acids, puffer fish, hybrids and hybrid triploids contained fatty acids such as SFAs, MUFAs, n-3 PUFAs and n-6 PUFAs. There were significantly different amounts of total fatty acids between groups (P<0.05), however, rates of changes in fatty acids did not differ significantly between groups (P>0.05). Gonadal hormone (estradiol and testosterone) changes in the river puffer and tiger puffer were significantly higher than that observed in hybrids and hybrid triploids. The hybrids and tiger puffers had higher amounts of growth hormone (thyroid stimulating hormone and thyroxine) than the hybrid triploids and river puffers (P<0.05).

Comparative Analysis of Tissue and Cell Cycle on the Far Eastern Catfish, Silurus asotus between Diploid and Triploid.

The influence of triploidization on histological characteristics of retina, trunk kidney, liver and midgut tissue, and cell cycle of tail fin and gill tissue in far eastern catfish, Silurus asotus were analyzed. In the infertile triploid fish, the nucleus and/or cell size of secondary proximal tubule cells of trunk kidney, hepatocyte and midgut epithelium are much larger than those of the corresponding cells in the diploid fish (P<0.05). However, triploid tissue showed fewer number of outer nuclear layer in retina and nuclei in secondary proximal tubule of trunk kidney than those for diploid tissue. The mean percentages of the Gl-, the S- and the G2+M-phase fractions were 92.5%, 3.2% and 4.3% in tail fin tissue of diploid, and 93.4%, 2.6% and 4.0% in those of triploid, respectively. There were no significant differences in the percentages of each cell cycle fraction between diploid and triploid. The mean percentages of each phase fractions were 75.1%, 11.1% and 13.8% in gill tissue of diploid and 85.2%, 8.9% and 5.9% in those of triploid, respectively. The differences of cell cycle between tail fin tissue and gill tissue were statistically significant in diploid and triploid (P<0.05). Also, the differences between diploid and triploid were statistically significant in tail fin tissue and gill tissue (P<0.05). Cyclin D1 and cyclin E expressions were not significantly difference between gill tissue and tail fin tissue, and protein expressions of induced triploid were higher than those of diploid. Results from this study suggest that some characteristics in the triploid exhibiting larger cell and nucleus size with fewer number of cell than diploid can be used as an indicator in the identification of triploidization and ploidy level in far eastern catfish.

Effects of Clove Oil and Lidocaine-HCl Anesthesia on Water Parameter during Simulated Transportation in the Marine Medaka, Oryzias dancena.

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.

Morphometric Characteristic between Diploid and Spontaneous Triploid Carp in Korea.

We used flowcytometry to ploidy verification after that investigate difference between diploid and spontaneous triploid through the truss dimension and classical dimension at crucian carp, Carassius auratus, crucian carp C. cuvieri and common carp, Cyprinus carpio collected from Hangang river, Hantangang river, Imjingang river, Geumgang river, Yeongsangang river and Nakdonggang river, Korea. There were significant differences among the three species for the truss dimensions anterior origin of dorsal fin (2)× anterior origin of anal fin (5), 2× anterior origin of pelvic fin (6), 2× origin of pectoral fin (7), posterior origin of dorsal fin (3)×5, 3×6, and 3×7 (P<0.05). There were no significant differences among the three species in the truss dimensions dorsal fin length (2×9) and eye diameter (ED)(P>0.05). On the other hand, there were no significant differences in the several classical dimensions of each species (P>0.05). Three classical dimensions, most anterior extension of the head (1)×2, 1×6 and 2× most posterior scale in lateral line (4) did not differ between the C. auratus diploid and spontaneous triploid (P>0.05). Two classical dimensions, 1×6 and longest length between most anterior extension of the head and gill cover (1×8) did not differ between the C. cuvieri diploid and spontaneous triploid (P>0.05). One classical dimensions, 1×2 did not differ between the common carp diploid and spontaneous triploid (P>0.05). There were significant differences in the each diploid and triploid species (P<0.05). These results suggest that the classification of each species and classification between diploid and spontaneous triploid morphometrical parameters used in this study are useful indices of morphometrical status in the each species from major river of Korea.

Effects of Cryoprotectants and Diluents on the Cryopreservation of Spermatozoa from Far Eastern Catfish, Silurus asotus.

The aim of this study was to compare the efficacy of cryopreservation methods for ex situ conservation of spermatozoa from far eastern catfish, Silurus asotus. The spermatozoa activity index (SAI) and hatching rates were higher in spermatozoa stored in Alserver's solution than those of spermatozoa stored in glucose solution. The SAI and hatching rates in all experimental groups gradually decreased with increasing duration of storage. Additionally, the SAI and hatching rates gradually decreased with increasing thawing temperatures at all storage durations (P<0.05). Based on the SAI and hatching rates, our results suggest that the optimal cryopreservation conditions of catfish spermatozoa involve storage in Alserver's solution with 15% ethylene glycol, and thawing at 25℃. Cryopreservation of spermatozoa is a useful and reliable technique for conserving gene resources and for artificial propagation of far eastern catfish.

Anesthetic Effect and Physiological Response in Olive Flounder (Paralichthys olivaceus) to Clove Oil in a Simulated Transport Experiment.

The optimum concentrations of clove oil as an anesthetic for olive flounder (Paralichthys olivaceus) and the stress response of the fish to clove oil anesthesia were determined over a range of water temperatures, and investigated in a simulated transport experiment using analysis of various water and physiological parameters. While the time for induction of anesthesia decreased significantly as both the concentration of clove oil and water temperature increased, the recovery time increased significantly (P<0.05). The plasma cortisol concentration in fish at each temperature increased significantly up to 12 h following exposure (P<0.05), then decreased to 48 h (P<0.05). The DO dissolved oxygen concentrations, pH values, and the fish respiratory frequencies decreased over 6 h following exposure to clove oil in all experimental groups (P<0.05), whereas the NH4+ and CO2concentrations in all experimental groups increased up to 6 h (P<0.05). The pH values and DO concentrations increased with increasing clove oil concentration (P<0.05) in the 6 h following exposure, and the CO2 and NH4+ concentrations and the respiratory frequencies decreased with increasing clove oil concentration (P<0.05). The results of this experiment suggest that clove oil reduced the metabolic activity of olive flounder, thus reducing NH4+ excretion and O2 consumption. In conclusion, clove oil appears to be a cost-effective and efficient anesthetic that is safe for use and non-toxic to the fish and users. Its use provides the potential for improved transportation of olive flounder.

Landmark-based Morphometric and Meristic Analysis of Serranidae.

The landmark-based morphometric and meristic analysis of the kelp grouper (Epinephelus bruneus), red spotted grouper (E. akaara) and seven-banded grouper (E. septemfasciatus) were performed to compare the differentiation of overall body shape and structure. The measurements of the morphometric dimensions were observed in 25 parts (truss dimension: 16 parts; head part dimension: 9 parts) of 38 morphometric dimensions and also meristic differences observed in 3 parts (dorsal fin, anal fin and caudal fin) of 6 meristic counts (P < 0.05). Observed morphometric characteristics primarily involved in truss and head part dimension, kelp grouper have larger values in caudal part of truss dimension, kelp grouper, red spotted grouper and seven-banded grouper have similar values in pectoral part of truss dimension, in addition to, results of head part dimension showed that red spotted grouper have smaller values in overall dimensions (P < 0.05). As meristic characteristics, kelp grouper have more number of anal fin rays than other fish, red spotted grouper have more number of dorsal soft rays than other fish, and seven spotted grouper have more number of anal soft rays, and caudal fin rays than other fish (P < 0.05). Photographed under the x-ray, kelp grouper have the most curved vertebral column and largest swim bladder than other fishes (P < 0.05). Our results of this study confirmed that 3 subfamily fishes adequately can distinguish with external body shape, and we hope that the results of our study could be used to identify in Serranidae family as taxonomical parameters.

Cytogenetic study of diploid and induced tetraploid in Korean rose bitterling, Rhodeus uyekii.

In this study, we induced tetraploidy in Korean rose bitterling, Rhodeus uyekii, by applying various hydrostatic pressure shock conditions. Tetraploidy was not induced under 4500 psi pressure treatment in any experimental group. Instead, the induction rate of tetraploidy was highest under 7500 psi hydrostatic pressure treatment. As a result, when the processing method was similar and as the process time increased, the induction rate of each experimental group increased; however, there was no significant difference (P > 0.05). The production rate was 3.1 %, which was highest in all experimental groups exposed to 6000 psi for 10 min after being fertilized for 100 min. The production rate was highest in the experimental groups treated with hydrostatic pressure alone, whereas the production rate was lowest in groups treated under hydrostatic pressure with chemical treatment. The abnormal rate of all experimental groups treated with 7500 psi for 20 min was very high, at about 5 %. Based on these studies, only hydrostatic pressure shock was considered effective at inducing tetraploidy based on the calculated hatching, abnormal, and induction rates. The most effective condition for inducing tetraploidy was 6000 psi of hydrostatic pressure shock for 10 min after being fertilized for 100 min. The chromosome number of the induced tetraploid Korean rose bitterling was 4n = 96, while that of the diploid was 2n = 48. In the diploid, there were 1 or 2 nucleoli in the cells, whereas the induced tetraploids contained 1, 2, 3, or 4. The DNA content of tetraploids and diploids were 3.68 ± 0.009 pg/nucleus and 1.84 ± 0.019 pg/nucleus, respectively, according to flow cytometric analysis. The DNA content and chromosome number of the tetraploids were twice that of the diploids.

Comparative Study of Growth and Gonad Maturation in Diploid and Triploid Marine Medaka, Oryzias dancena.

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.

Morphometric Characteristics and Fin Dimorphism between Male and Female on the Marine Medaka, Oryzias dancena.

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.