We investigated the change mRNA expression of GtHs subunits (FSHβ, LHβ) in the pituitary, androgen receptor (AR), estrogen receptor (ERα) in gonad and histological observation of gonads in longthooth grouper Epinephelus bruneus by treatment Femara, an aromatase inhibitor (AI). Longtooth grouper (body weight 408±43.1 g; one year) cultured in Future Aquaculture Research Center, NFRDI were used in the experiments. The experiment was conducted for 12 weeks from 21 August 2013. Fish received intramuscular injection of AI at 5 mg/g BW dose in three times every 3 weeks. Fish were sampled pituitary and gonads at 3, 6, 12 weeks post-injection (n=50). The mRNA levels of FSH-β, LH-β in pituitary and AR, ERα mRNA in gonad were evaluated using qRT-PCR and qPCR. The histological change of gonads observed on light microscope. The gonads of control group contained most perinucleolus oocyte. At 3 to 6 weeks post-injection, the gonads of AI-treated group contained a few degenerated oocytes, spermatogonia and spermatocytes. At 12 weeks post-injection, gonads contained spermatids undergoing spermatogenesis. From 6 to 12 weeks post-injection, the expression level of GtHs subunits mRNA in pituitary was significantly higher than control group. The expression level of AR mRNA in gonad was higher than control group from 3 to 12 weeks post-injection. The expression level of ERα mRNA in gonad was lower than control group from 6 to 12 weeks post-injection. These results suggest that immature longtooth grouper with AI treatment induced masculinization via change of GtH subunits in pituitary, AR and ERα mRNA in gonad.

This study was investigated spawning behavior, structure of egg masses and egg development in Aplysia kurodai inhabiting the coastal waters of Jeju Island, Korea. The mating and courtship behavior of A. kurodai occurred in the form of unilateral copulating with chain formation. In chain copulation, only the first animal acted as a female; the second and succeeding animals acted as males (sperm donors) to the animals in front and as females to the animals behind. The fertilized eggs were packaged in capsules that are embedded in jelly to form a cylindrical string called an egg masses. The number of capsule per cm of the egg masses was 55 to 60 capsules and each capsule within the egg masses held 15 to 25 eggs. After spawning, the egg masses were bright yellow or orange in color. This egg masses color not changed until embryos developed into trochophore stage. Thereafter, as embryo developed from trochophore stage to veliger stage the egg masses color became brownish. The fertilized eggs were spherical, with a diameter of approximately 80±1 μm at spawning. At 5 to 6 days after spawning, the embryo developed into trochophore stage and began to rotate within the egg capsule. In the trochophore stage, the precursor of the velum, called the prototroch or prevelum, developed. At 10 days after spawning, the prevelum is transformed into the velum, and the trochophore developed into veliger stage. Between 10 to 15 days after spawning, the veligers broke out of the egg capsule, and hatched as free-swimming larvae.

The fertilized eggs of E. septemfasciatus are spherical and transparent with buoyancy at 790 to 890 μm (average 821.8±2.0 μm) in diameter with 170 to 230 μm oil globules (average 192.9±0.93 μm). Hatching began approximately 46 and 35 hours after fertilization at 22.0℃ and 25.0℃ water temperature, respectively. The average total length of newly hatched larvae was 1.75±0.03 mm. Most of the yolk and oil globules were absorbed within 3 to 4 days after hatching. The larvae reached 2.48 to 2.72 mm in total length, and their mouths and anuses opened at 3 to 4 days after hatching. In this time, the mouth diameters of the larvae were 0.209 to 0.238 mm. The larvae reached 3.24 to 4.15 mm in total length at 11 to 17 days after hatching, and began to metamorphose at the time the second dorsal and pelvic spines appeared and elongated. The abdominal cavity was densely lined with melanophores. The larvae reached 5.12 mm in total length at 24 days after hatching.

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: 99.5±0.8%, DMSO 7.5%: 99.5±0.7%, and DMSO 10.0%: 99.6±0.6%). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: 96.2±2.3%, DMSO 7.5%: 95.3±3.6%, 10.0%: 96.6±1.8%) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm (98.4%±0.5) and DMSO concentration level of 5.0% (97.8±0.1%) were not significantly different. However, with 7.5% (97.2±0.6%) and 10.0% DMSO concentrations (95.9±0.2%) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; 66.8±1.8%, HR: 82.0±12.9%) and 2 years (FR; 78.5±14.8%, HR: 79.3±0.6%) cryopreserved sperm were lower than control (FR; 100%, HR: 91.1±3.6%) and 2 days cryopreserved sperm (FR; 99.6±0.6%, HR: 96.6±1.8%). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

To effects of sex maturation in olive flounder by regulating long photoperiod, gonadal development and GTH mRNA expression in the pituitary were investigated. Photoperiod was treated natural photoperiod and long photoperiod (15L:9D) conditions from September 2011 to March 2012. The results showed that natural photoperiodic group showed a higher gonadosomatic index (GSI) than long photoperiodic group during the spawning season (March 2012). The histological analysis of ovarian tissue showed that natural photoperiod group of ovaries contained vitellogenic oocytes, but long photoperiod group of ovaries mainly contained perinucleolus staged oocyte and oil-drop staged oocytes. The FSH mRNA of olive flounder, under natural photoperiod group, showed a significantly higher expression but no significant difference under long photoperiod group. The LHβ mRNA showed a significantly higher expression only under natural photoperiod group. These results may suggest that long photoperiodic information regulates secretion of pituitary FSH and LH and maintain early growing stage of gonadal development in this species.

To understand the sex reversal characteristics in the longtooth grouper (Epinephelus bruneus), this study examined the sex reversal and artificial masculinization of wild caught E. bruneus reared in indoor rearing tank after a 17α-methyltestosterone injection. To domesticate a broodstock, 64 wild caught E. bruneus, between 47.0 to 110.0 cm in total length and from 1.5 to 21.4 kg in body weight, were reared in indoor rearing tank (4.0 to 5.0 m wide, and 2.5 to 3.0 m depth) for four years. Seven specimens showed sex reversal from female to male during indoor rearing condition, whose total length and body weights were from 63.0 to 99.0 cm and from 4.4 to 13.2 kg, respectively. After inducing artificial masculinization in 14 female E. bruneus with a 17α-methyltestosterone (2.0 mg/kg BW) implants for 3 years, spermiation occurred in 9 specimens (total length: 54.0 to 68.0 cm, body weight: 2.3 to 4.3 kg). Among the female to male sex reversals, two specimens returned back to being female, whose body weights were 2.8 kg (initially 2.6 kg) and 2.7 kg (initially 2.3 kg). Therefore, this study suggested that E. bruneus (> 3.0 kg) was more effective in masculinizing by 17α-methyltestosterone implants.

Gonadal development and reproductive cycle of Aplysia kurodai inhabiting the coastal waters of Jeju Island, Korea were investigated based on monthly changes of gonadosomatic index, gametogenesis, and developmental phases of ovotestis. A. kurodai was simultaneous hermaphrodite; the ovotestis generally embedded in the posterior dorsal surface of the brownish digestive gland. The ovotestis is composed of a large number of follicles, and both oocytes and sperm are produced in the same follicles. In the sampling periods, the adult A. kurodai population have characteristic of seasonal pattern present during only 10 months. The reproductive cycle can be grouped into the following successive stages in the ovary: inactive (December to February), active (December to April), mature and spawning (April to September). The gonadal development of A. kurodai coincided with rising temperature, and spawning occurred from April to September, when the temperature was high. The histological observations of the ovotestis suggested that this species have a single spawning season that extend over six months.

Light characteristics are very specific in the aquatic environment. Fish vision and different light spectra perception are related to each species’ natural habit. Light is one of the main environmental conditions and can be easily manipulated in artificial rearing settings. Cholecystokinin (CCK) and mucus-secreting goblet cells are the main regulators of digestion. In this study, we established whether the light spectrum (natural condition, full spectrum: green, 520 nm; red, 590 nm, and blue, 480 nm) influences growth performance and digestive activity related to CCK mRNA expression and mucus-secreting goblet cell activity in order to develop a good management protocol and optimal rearing system for the longtooth grouper. For each light spectrum, fish were reared 12 weeks under a flow-through system and fed commercial pellet diets once daily. At the end of the experiment, the final body weights differed among the fish reared under different light spectra. The highest growth performance value was observed in fish reared under the green light condition. On the other hand, the growth performances of fish in the natural and blue light conditions were drastically decreased in last 3 weeks of the experiment. CCK mRNA expression and mucus-secreting goblet cell activity were significantly higher in the fish under green light condition than in the fish under the natural, red, and blue light conditions. Rearing of the longtooth grouper under the green light condition had positive effects on fish growth performance and digestion. We recommend that the appropriate light spectrum for the artificial culture of the longtooth grouper is the green light condition from the perspective of growth performance and the synergistic effects of CCK and mucus-secreting goblet cells. However, longer light treatment periods are needed in future investigations to clarify the effects of light spectrum on the longtooth grouper. Together with the findings of the present study, such studies would result in better understanding of the digestive physiology and contribute to the development of optimal rearing management for commercial production of the longtooth grouper.

제주도 옹포와 법환 연안에 서식하는 분홍성게의 생식소 발달과 생식주기를 조사하기 위하여 1994년 12월부터 1995년 12월까지 채집된 개체를 대상으로 생식소중량지수(GSI)의 월별 변화, 월별 생식소 발달 단계를 조사하였다. 옹포와 법환 지역에 서식하는 분홍성게의 생식소중량지수의 월별 변화는 유사하였다. 암컷은 옹포와 법환 모두 12월에 각각 17.8± 4.04, 13.8±1.51로 최고값을 보였고, 수컷도 옹포와 법환 모두 12월에 각각 15.4±1.53, 13.6±1.32로 최고값을 나타냈다. 주 산란은 옹포와 법환 수온과 일장 주기가 감소하는 11월～2월에 일어났다. 생식소의 조직학적 관찰 결과, 제주도 옹포와 법환 연안에 서식하는 분홍성게의 암ㆍ수 배우자세포 발달 양상은 동시 발달형이고, 년 1회의 산란시기를 갖은 것으로 생각된다.

The transcription factors, DMRT1 and FOXL2, play a role in fish sex differentiation of the bipotential precursor into the male and female pathway, respectively. In order to provide the molecular background for understanding hormonal regulation in sexual determination and differentiation in the red-spotted grouper, Epinephelus akaara, one of commercially important epinephelines, and is often used to study protogynous sex change. First, we amplified the partial sequence of two genes (DMRT1 and FOXL2) from the gonad of red-spotted grouper. Also, we surveyed the tissue-specific and sex-specific expression pattern of each genes by RT-PCR. The effects of 17α-methyltestosterone (MT) in the sexually immature gonad of red-spotted grouper were investigated by Real-time quantitative RT-PCR. Fish were treated with MT-dipping method from around 70 days after hatching (DAH) for two month. DMRT1 and FOXL2 cDNA flagments consist of 489 and 836 base pairs (bp) and encodes a protein of 162 and 278 amino acids, respectively. RT-PCR revealed that DMRT1 mRNA was expressed higher level in the testis. Foxl2 was expressed extensively in the neural and peripheral tissues with its highest level in the ovary, indicating a potential role for Foxl2 in the brain-pituitary-gonad axis. Real-time quantitative RT-PCR analyses showed that DMRT1 mRNA expression was upregulated in the MT-treated fish. These results suggest that the sex inversion of red-spotted grouper by MT might be due to the suppression of FOXL2 gene expression, and resulting in the induction of the 11-KT secretion.

We investigated the androgenic effects of 11-ketotestosterone (11-KT) on gonadal sex reversal and spermatogenesis in honeycomb grouper Epinephelus merra by method of gonadal biopsy. 11-KT was injected intramuscularly at a concentration of 1 and body weight. The proportion of cross sectional area of the gonad occupied by each germ cell type was measured and compared pre- and post-injection group. During the sex change phase, the distribution ratio of oocytes was decreased in all fish of 11-KT treatment group while the distribution ratio of spermatocytes was increased than pre-injection group. In male phase, all fish of 11-KT treatment group shown the increased distribution ratio of spermatocytes, but the distribution ratio of spermatozoa was decreased than pre-injection group. The present results suggest that 11-KT can stimulate degeneration of oocytes, proliferation of spermatocytes and spermiation in honeycomb grouper.

The surface sediments inside Songsanpo and Seogwipo Harbors, major harbors of Jeju Island, were collected three times (June, September and December, 2001) and analyzed for 16 polycyclic aromatic hydrocarbons (PAHs) recommended by US-EPA as priority pollutants to assess their distribution levels and their biological effects on the marine organisms, and to suggest their possible origins. The concentrations of total PAHs inside Songsanpo and Seogwipo Harbors ranged from 41 to 288 ng/g on a dry weight basis with a mean value of 121 ng/g and from 14 to 268 ng/g with a mean value of 119 ng/g, respectively, and the levels were low as compared with those in other areas of the world. The sedimentary PAHs may be correlated with organic carbon and mud content to some extent. Based on comparisons of individual and total concentrations with effects-based sediment quality guidelines, the potential for the biological effects on the marine organisms were expected to be very low. From the examinations of the four PAH origin indices, such as LMW/HMW (low molecular weight 2-3 ring PAHs over high molecular weight 4-6 ring PAHs), phenanthrene/anthracene ratio, fluoranthene/pyrene ratio, chrysene/benzo[a]anthracene ratio, it can be concluded that the sediment PAH contaminations were ascribed to both of pyrolytic and petrogenic origins.