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Artificial Spawning Behavior and Development of Eggs, Larvae and Juveniles of the Red Spotted Grouper, Epinephelus akaara in Korea

This study was conducted in order to examine the egg development in red spotted grouper, Epinephelus akaaraand 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.


INTRODUCTION
Studies of early stages in the life cycle of fish are performed to obtain information such as morphological and physiological characteristics of eggs, as well as speciesspecific characteristics and developmental traits that can be observed during embryogenesis and early development. These studies provide taxonomical, developmental, and ecological knowledge, which can be used for the preservation of fish resources and for increasing their proliferation through seed production in different ways (Song & Choi, 2000).
Epinephelus akaara is a fish that belongs to the Perciformes order and Serranidae family, with 12 genera and 27 species found in Korea. Epinephelus akaara is distributed in the south coast of Korea, Jeju-do, Japan, and China (Kim et al., 2005). The Serranidae are currently an economically important taxonomic group, with high industrial value, and several studies on the technical development of their stock production have been done (Heemstra & Randall, 1993;Froese & Pauly, 2014).
Domestic studies of the Serranidae include studies that have been performed on maturation and sex-reversal of the red spotted grouper, Epinephelus akaara ; on the live food habits , time of feeding , changes of egg quality , and early life cycle of the sevenband grouper, Epinephelus septemfasciatus (Park et al., 2014). Besides these, studies on the skeletal , and egg development; and the effects of water temperature and salinity (Yang et al., 2007;Cho et al., 2015) have also been conducted. Overseas studies include studies on egg development and on morphological development of larvae and juveniles of the sevenband grouper, Epinephelus septemfasciatus (Kitajima et al., 1991), that included studies on deformities in larvae and juveniles (Nagano et al., 2007), their spawning habits, and early life cycle (Ukawa & Higuchi, 1966).
Studies on early life history are very useful for distinguishing between similar fish species, and for determining their relationships. A number of developmental traits provide significant data for elucidating taxonomic relationships (Blaxter, 1974;Balon, 1985). Epinephelus akaara is known to have a characteristic extension of rays in the dorsal and ventral fins at the early larval stage (Ukawa & Higuchi, 1966;Song & Noh, 1998). Although it is easily distinguished from larvae and juveniles of other marine fish, its morphology is similar to that of other Serranidae family larvae, which makes species identification of this taxonomic group difficult. Therefore, this study aims to provide information that might be useful for taxonomic research by investigating egg development, and the morphological development of larvae and juveniles of Epinephelus akaara.

MATERIALS AND METHODS
1. Breeding of adult fish and inducing spawning behavior Fifty adult Epinephelus akaara fish (20 females and 30 males) were used in this study. They matured in June 2013 (37.9±2.99 cm in total length and 888.7±244.7 g in weight).
These fish were selected from 200 fish caught from the coast of Geomun-Island, Yeosu-si, Jeollanam-do using fish trap in September 2012 and grown in a fish holder (7×7×5 m). For the feed, combination of horse mackerel, squid, and oyster was provided twice a day at 1-2% of fish weight.
To induce spawning behavior, 100 µg/kg of LHRHa was injected into the fish on the dorsal muscle of the lower side of the first ray and the fish were kept in a small-sized fish holder (2×2×2 m).

Egg development
Fertilized eggs were kept in a 1,000 mL glass beaker and the temperature of breeding water was maintained at 22.0-23.0°C (mean 22.5°C) and salinity at 32.5-33.5% (mean 33.0%) until hatching. Half the breeding water was replaced 5 times each day. Thirty eggs were retrieved randomly and their size was measured with a scale of 0.01 mm, using a phase-contrast microscope (Leica DE DM750, Germany). Egg development was monitored under a stereoscopic microscope (Nikon SMZ18, Japan) and photographs were also taken.

Morphological development of larvae and juveniles
Newly hatched larvae were kept in a circular water tank made of polypropylene (PP) and grown using the intensive pond system under mild ventilation with aeration and liquefied oxygen. As food supply for larvae, 5-10 Brachionus rotundiformis per mL were provided for 30 days after yolk absorption, and they were supplemented every 6 h to maintain the density. Combination of Brachionus rotundiformis and Artemia sp. were provided from day 30 to 50, and assorted feed (Lovelarva, Japan) was provided after day 50. For studying the morphological development of larvae and juveniles, 10 individuals were retrieved immediately post hatching and anesthetized using an anesthetic (MS-222, Ethyl 3-aminobenzoate methanesulfonate, Sigma Aldrich Co., St. Louis, USA), after which each region of the fish body was measured at the scale of 0.01 mm and observed under a stereoscopic microscope. Morphological developmental stages of larvae and juveniles were classified according to Kendall et al. (1984).

Spawning behavior
The spawning behavior of Epinephelus akaara was divided into 4 stages as shown in Fig. 1. During the spawning period, males showed nuptial coloration on their head in a spotted pattern, which became fainter towards the tail.
Females had an inflated abdomen and the area around the gonopore was red and protruded. Males chased after females  1C). After spawning, males and females were separated and initiated spawning again (Fig. 1D).
on the tail (Fig. 2B). At the time point 25 h post-fertilization, the embryo started moving actively, the heart had formed, and the heart beat at this stage with the rate of 65-72 beats per min (Fig. 2C). Hatching began at 27 h postfertilization, as the embryo broke out of the egg membrane with its tail first ( Fig. 2D-E).

Morphological development of larvae and juveniles
Newly hatched pre-larvae were 2.02-2.17 mm in size Post-larvae at 9 days post hatching were 3.30-3.58 mm in length (mean 3.44±0.20 mm) and 6 rays were formed on the round pectoral fin, while 1 ray was formed on the dorsal fin. Branch-shaped black chromophores, which were Post-larvae at 15 days after hatching were 3.88-4.07 mm in length (mean 3.98±0.13 mm), and 1 ray that had formed on the dorsal fin was extended. Two rays were formed on the ventral fin and 8 rays on the caudal fin. The membrane fin was connected from the dorsal fin ray to the anus. Black chromophores were deposited on the caudal peduncle and also at the end of the dorsal and ventral fin rays in a spotted pattern (Fig. 4A).
Post-larvae at 55 d after hatching were 31.9-35.2 mm in total length (mean 33.6±2.33 mm). Red color was deposited on the entire body and black chromophores were deposited in a spotted pattern. As the number of rays in the ventral fin increased to 8, the number of fin rays, body color, and morphology was similar to that in the adult fish, which led to the juvenile stage. At this stage, the position of the anus was at 49.4% of the body length, proximal to the center of the body (Fig. 4E).

DISCUSSION
Fertilized eggs of Epinephelus akaara are globular-shaped, colorless, and transparent free pelagic eggs with one oil globule. Other marine fish that have free pelagic eggs include Paralichthys olivaceus (Han & Kim, 1997), E.
bruneus (Lee & Go, 2003), E. septemfasciatus (Park et al., 2014), Oplegnathus punctatus , O. fasciatus (Koh & Kim, 1992), Acanthopagrus schlegelii (Kim, 1970) and Scomber japonicus , Water temperature is an environmental factor that affects the development of larvae and juveniles, determines the hatching time for fertilized eggs, and directly affects organogenesis from the embryological aspect (Hempel, 1979;Byun et al., 2007 Differences in time at similar water temperature are most likely a characteristic of the species, but most showed a close relationship with water temperature. Such tendency is also observed in various kinds of fish including Girella punctate and G. melanichthys (Oh et al., 2010), halibut , Gadus macrocephalus (Lee et al., 2007), mackerel (Hwang et al., 2008), Takifugu pardalis (Han & Cho, 2007), and Microstomus achne (Byun et al., 2009 al., 1992), which were all shorter than the length of Epinephelus akaara larvae. Length of E. bruneus larvae was 2.02 mm (Lee & Go, 2003), which was in a similar range.
Fish of the Serranidae family undergo morphological changes at the post-larval stage. The onset of these changes depends on the time when the second ray of the dorsal fin and the first ray of the ventral fin begin to extend. In the present study, the onset of morphological changes in Epinephelus akaara was observed to be on the 15th day after hatching, and when the length was 3.98 mm. Similar results were reported by Ukawa & Higuchi (1966), where the onset of morphological changes were observed 15 days after hatching and when the length was 4.05 mm and by Song & Noh (1998)