This study was conducted in order to examine the egg development in red spotted grouper, Epinephelus akaara and the morphological development of its larvae and juveniles, and to obtain data for taxonomic research. This study was conducted in June 2013, and 50 male and female fish were used for the study. One hundred μg/kg of LHRHa was injected into the body of the fish for inducing spawning, and the fish were kept in a small-sized fish holder (2×2×2 m). Eggs were colorless transparent free pelagic eggs, 0.71–0.77 mm large (mean 0.74±0.02 mm, n=30), and had an oil globule. Hatching started within 27 h after fertilization. Pre-larvae that emerged just after hatching were 2.02–2.17 mm in total length (mean 2.10±0.11 mm), their mouth and anus were not opened yet, and the whole body was covered with a membrane fin. Post-larvae that emerged 15 days post hatching were 3.88–4.07 mm in total length (mean 3.98±0.13 mm), and had a ventral fin with two rays and a caudal fin with eight rays. Juveniles that were formed at 55 d post hatching, were 31.9–35.2 mm in total length (mean 33.6±2.33 mm), with red color deposited over the entire body, and black chromophores deposited in a spotted pattern. The number of fin rays, body color, and shape were the same as that in the adult fish.

Red spotted grouper, Epinephelus akaara is a popular aquaculture species in many Asian countries. This species is a protogynous hermaphrodite that first differentiates into female and changes to male later. Due to this reproductive characteristic, stable supply of male and female gametes is a key to the success of seed production in this species. Thus, understanding early gonadal differentiation is required to develop effective sex control techniques. Red spotted grouper were reared in indoor tanks and sampled every 5 days from 40 days post-hatch (DPH) to 130 DPH. Changes of gonadal tissues were examined and analyzed by means of histology. A pair of gonadal primordium has already existed underneath the kidney in the posterior part of the body cavity at 38 DPH when this study began. Gonadal primordia of 38, 40 DPH consisted of germ cells surrounded by a few somatic cells. The blood vessel was observed in the gonadal primordium at 45 DPH. The number of somatic cells and size of gonadal primordium increased age-dependently up to 60 DPH. Formation of ovarian cavity was obvious by two protuberant aggregations of somatic cells at 65 DPH. Completed ovarian cavity and oogonia were first observed in the gonad of one fish sample at 105 DPH. Based on these histological observations, it can be suggested that induction of primary male differentiation could be more successfully applied at around 60 DPH in this species.

We sought to identify optimal temperatures for aquaculture of juveniles at a length of 7.2 cm ± 0.1 cm. The impact of four temperatures (20℃, 24℃, 28℃, and 32℃) on feeding rate, growth performance, and stress responses (i.e., blood cortisol, glucose, alanine transaminase [ALT], and aspartate transaminase [AST] content) of juvenile red-spotted grouper was measured over a period of 6 wks. Red-spotted grouper had significantly higher final body weight (g) at 24℃ and 28℃ than at 20℃ or 32℃, and greater percentage body weight increase/day at 24℃ than at 28℃. There were significant differences in feeding rate, depending on the number of weeks. The 6-wk feeding rate was highest at 28℃, followed by 32℃, 24℃, and 20℃, while the lowest feed conversion ratio occurred at 24℃, followed by 28℃, 32℃, and 20℃. After 6 wks, there were no significant differences in plasma cortisol or AST levels. However, there were significant increases in plasma glucose and ALT levels at 32℃ compared to at 20℃. Although red-spotted grouper at 32℃ did not show a significant increase in blood cortisol levels, our results suggested that at 32℃ the red-spotted grouper experiences high energy-demand (i.e., high glucose level) and stress (i.e., high ALT), especially in the liver. Based on body weight, % body weight increase/day, feed conversion ratio, and stress indicators, the optimal temperature for aquaculture of red-spotted grouper was ~24℃. Additional studies at temperatures between 24℃ and 28℃ will be required to identify the exact optimal temperature for juvenile red-spotted grouper culture.

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. 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 mucus-secreting goblet cell activity in order to develop a good management protocol and optimal rearing system for nursery stage of Epinephelus akaara. For each light spectrum, fish (11.5 ± 0.2 g in mean initial body weight, 9.0 ± 0.1 cm mean initial total length) were reared 16 weeks under a flow-through system and fed commercial pellet diets twice 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 and feed efficiency were observed in fish reared under the green light condition. Mucus-secreting goblet cell activity was significantly higher in the fish under green light condition than in the fish under the natural, red, and blue light conditions. Rearing of E. akaara under the green light condition had positive effects on fish growth performance and digestion. We recommend that the appropriate light spectrum for nursery stage of E. akaara is the green light condition from the perspective of growth performance and the synergistic effects of mucus-secreting goblet cells. However, longer light treatment periods are needed in future investigations to clarify the effects of light spectrum on each growing stage of E. akaara.

Red spotted grouper (Epinephelus akaara) is one of the most popular and important grouper species for aquaculture in South-East and East Asia thanks to its fast growth and high market value. This species is known as a protogynous hermaphrodite which first differentiates into females and changes to males later. Natural sex change in this species occurs at 2 or 3 years of age. Many studies have been conducted, so far, to develop standardized methods for artificial sex reversal by treatment with sex steroid hormones (Bhandari et al., 2005; Lee et al., 2014). However, sex-changed male groupers showed testes with ovarian cavity, and their sperm production was very low. Induction of primary sex differentiation directly into males would be an alternative approach. Identification of the exact primary sex differentiation period is the prerequisite for this approach. Red spotted grouper were reared in indoor tanks and acclimatized at 25±2℃ under natural photoperiod. Fish were sampled at 60 days after hatching. The samples were processed for histological analysis using standard techniques in an automatic tissue processor, sectioned at 8 μm in thickness and stained with haematoxylin and eosin. The result of this study shows the morphological characteristics of gonadal primordium, and suggests that the timing of gonadal differentiation in red spotted grouper is at least 10 days earlier than the previous study of Lee et al. (2014) which observed gonadal primordium at 70 days after hatching.

This study was conducted to determine the stress response [ethological (operculum movement number (OMN)), hematological (hematocrit and hemoglobin), biochemical (glucose, cortisol and glutamic oxaloacetic transaminase (GOT))] in red spotted grouper, Epinephelus akaara during exposure of different water temperature in winter season. This species (Total length, 18.56±0.34 cm) previously maintained in water temperature of 15°C were transferred to 15, 20 and 25°C. During experimental period (7 days), OMN, hematocrit (Ht), glucose and GOT values were significantly high in 15°C when compared to 20 and 25°C. Hemoglobin value was also increased at 15°C, but no significant differences. There was no differences in cortisol levels among the temperature groups. No fish mortality was observed during the experimental period. From these results, 15°C is likely more stressful to red spotted grouper than 20°C and 25°C. These observations confirm that red spotted grouper adapts better to temperatures between 20 and 25°C during the winter season.

We investigated the androgenic effects of 17α-methyltestosterone (MT) on gonadal sex reversal in juvenile red spotted grouper Epinephelus akaara. The fish were immersed in 17α-MT at 1 and 5 mg/L. Treatment method of 17α-MT was once weekly for 4 and 8 weeks. Fish were sampled at 12 months after end of the treatment period in order to histological analysis. At the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 DAH in red spotted grouper. At 12 months after end of the treatment period, control group, 17α-MT 1 mg/L treatment group for 4 and 8 weeks, and 17α-MT 5 mg/L treatment group for 4 weeks were all female. However, sex-changed males without ovarian cavity were observed in the 17α-MT 5 mg/L treatment group for 8 weeks. In grouper, we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

Skeletal deformities are important factor of evaluation of fish value commercially. Deformities of opercular are commonly observed type of fish deformation. Although these malformations in fish can be caused by culture conditions, the environmental factors are unknown. This study examined the effect of water temperature on the opercular deformity of the red spotted grouper, Epinephelus akaara. Experimental fish (TL; 7.49±0.10 cm) were respectively divided into 3 groups that were reared at 20, 24, 28℃ for 6 weeks. All specimens were photographed from the left lateral view using a Canon EOS 70D. We placed 11 landmark points for visualization the shape differences of operculum in the whole body. In order to measure the reduction of opercular, we estimated total length (TL) and shortening of the distal part (distance between landmarks 10 and 11). After 6 weeks, both growth rate and incidence of opercular shortening were high in 24 and 28℃. At 28℃, the distance of distal part of operculum was the highest as 0.36 cm and exposure to the 24℃ induced the highest growth rate during this experiment. On the other hand, both growth rate and opercular deformity were low at the lower temperature (20℃). This study shows opercular malformation as well as the growth rate of E. akaara are influenced by the high water temperature.

As a preliminary investigation into the effect of environmental factors control for gonadal development, we examined the involvement of photoperiod and water temperature in ovarian development of Epinephelus. akaara. For the induction of sexual maturation, E. akaara reared in recirculating aquaculture system (RAS). During November 2013, the photoperiod and water temperature was adjusted to 12L:12D and 18℃, respectively. In the photo-thermal treatment group, every 3 weeks daylight was increased as follows a 13L:11D and 14L:10D, and control group was maintained under natural condition. After 9 weeks, water temperature was increased 23℃ in photo-thermal treatment group. The sampled fish every 3 weeks revealed increase in gonadosomatic index (GSI; 5.18±1.38), oocyte diameter and vitellogenic oocytes (423.9±36.1 ㎛) were observed in gonads 12 weeks under photo-thermal treatment group. However, ovarian development was maintained immature stage in control group. In this environmental factors manipulation trial, seventy one of the 95 females (578.4 ± 25.4 g in mean body weight, 31.0 ± 0.5 cm mean total length) treated with HCG injection (doses 500 IU/kg BW) were induced ovulation by artificial stripping. The total volume of ovulated eggs were 3,470 ml and the total volume of fertilized eggs was 3,295 ml. The fertilization rate and hatching rate were 95% and 98%, respectively. These results suggest that the photoperiod as well as water temperature are major environmental factors in triggering the gonadal development of E. akaara.

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.