SUISANZOSHOKU 51(3), 295-306 (2003) Morphological Development of Embryos and Juveniles in the Mozambique Tilapia, Oreochromis mossambicus as a Direct Developmental Fish under Rearing Conditions Katsunori TACHIHARA*1,2 and Emi OBARA*1 (Accepted June 17, 2003) Abstract: The Mozambique tilapia, Oreochromis mossambicus is one of the species introduced to Okinawa Island. This species breeds widely in the rivers of the island. In order to understand the biological aspects of O. mossambicus under the natural conditions of the island, a detailed observa- tion of early morphological development was done under rearing conditions. The development of O . mossambicus was studied; it consisted of an embryonic phase of approximately 88 hours and 30 minutes, a free embryonic phase of about 12 days, followed by the juvenile period. The embryo became free-swimming on the sixth day of the free embryonic phase. The osteological develop- ment using enzyme-cleared specimens indicated that after reaching the juvenile stage, develop- ment of almost basic bones was complete 25 days after hatching. Key words: Oreochromis mossambicus; Direct development; Early life history; Okinawa Island The Mozambique tilapia, Oreochromis mos- sambicus is a freshwater fish, which has been Materials and Methods introduced to Okinawa Island1-3). The species is endemic to southern African rivers, lagoons Three mouthbreeding females (122.7 to and lakes, and ranges from the lower Zambezi 124.7 mm in standard length) harboring newly system in the north to the Bushmans River fertilized ova were collected from the Onaha in the south4). This species was introduced to River, Nishihara, Okinawa, Japan, on June 4, Okinawa Island in 1954 from Taiwan as a source 1997, using a cast net (mesh size: 1•~1 cm). of cheap protein5,6). Subsequently, some fish They were brought to the laboratory and each escaped into natural rivers and have recently individual was measured for the standard been found to be breeding widely in the wild length (SL), using a caliper to nearest 0.1 mm, on the islands. Despite the fact that the spe- and weighed with a digital balance to the near- cies is one of the dominant freshwater fishes in est 0.01 g. Subsequently, they were reared sepa- Okinawan rivers, to date only limited research rately in 10 liter polycarbonate tanks containing on fundamental biological aspects has been con- fresh water, under environmental temperature ducted2). To understand the aspects of the spe- of 26•}0.8•Ž with a normal photoperiod. Several cies naturalization to this island's environment, ova were removed from the buccal cavity of a detailed observation of early morphological the females and reared in 500 ml beakers and osteological developments was conducted. aerated with an air stone, under the same previ- ously described conditions. The ovum, which *1 Laboratory of Fisheries Science & Coral Reef Studies , Faculty of Science, University of the Ryukyus, Senbaru, Nishihara, Nakagami, Okinawa 903-0213, Japan. *2 Corresponding author: Tel/Fax: 098 -(895)-8556 , -8576; E-mail: [email protected],ac.jp 296 K. Tachihara and E. Obara remained in the females' mouths, were used as 1D). Formation of the embryo was visualized a control for the ovum, which had been trans- at 22 hours and 27 minutes after the first obser- ferred to the beakers. After hatching, two to vation. At the posterior tip of the embryo, the four embryos of each clutch were fixed in 5% Kupffer's vesicle appeared ventrally (Fig. 1E). formalin every morning. From the second to At 26 hours and 17 minutes after the first third day after hatching, the embryos were fed observation, the notochord and nine somites with a small amount of newly hatched Artemia had formed. Several melanophores were con- nauplii and compound fish food in an amount spicuous in the yolk sac (Fig. 1F). At 28 hours, that appeared to be adequate (small residue formation of the optic vesicle was noted, the remaining on the bottom of the tank). number of somites had increased to 17, and Observations, measurements, and sketches the Kupffer's vesicles had disappeared (Fig. of the embryos and juveniles were made using 1G). At 48 hours and 15 minutes after the first live and fixed specimens under a dissecting observation, the following was noted: the optic microscope containing an ocular scale of 10:100 lenses and the auditory vesicles had formed; and a profile projector (Nikon, V-12B) with the heart was present, and in the ventral por- magnification lenses of 20x and 50x. tion; the number of somites had reached 21; Using 36 free embryo and juvenile samples and melanophores had somewhat increased in preserved in 5% formalin, preparation of the yolk sac (Fig. 1H). At 56 hours and 10 min- enzyme-cleared specimens was done according utes after the first observation, the following to the method of Dingerkus and Uhler7). The was noted: the head had grown with the for- nomenclature of cartilaginous and ossified ele- mation of a rudimentary brain; the number of ments was primarily derived from that of Ueno8) somites had increased to 28; and the posterior and Fujita9). extremity of the embryo had detached from the yolk sac (Fig. 11). At 74 hours and 40 minutes Results after the first observation, the auditory vesicles appeared, and the embryo was almost com- Ovum development pletely occupying the perivitelline space (Fig. The ovum shape of O. mossambicus was 1J). Hatching occurred during 85 hours and ovoid, with the longitudinal axis longer than 15 min to 88 hours and 30 minutes after the first the transverse axis (2.72•}0.10 mm and 1.96•} observation; it began with frantic motions of the 0.08 mm, n=30) and the yolk was homoge- posterior caudal extremity neous with no oil globules. Newly collected ova were brought to the laboratory and the first Free embryonic development observation was made 1 hour and 45 minutes Newly hatched free embryos contained after sampling. Organogenesis had begun and a large yolk sac, and the notochord length was found to be in the blastocyte stage (Fig. (NL) was 4.0 mm. Melanophores were pres- 1A). The morula stage was reached 3 hours ent adjacent to the free embryonic body (Fig. and 30 minutes after the first observation; the 2A). One day after hatching, the following was blastodisc developed following a synchronous noted: the NL was 4.5 mm; eye pigmentation cleavage with an increase in the number of the had begun; and the yolk sac had assumed a blastomeres (Fig. 1B). At 4 hours and 10 min- slightly inclined position (Fig. 2B). At two days utes after the first observation, the embryos of age, the following was noted: the NL was had reached the blasutula stage with the blas- 5.6 mm; the mouth had opened; the movement tomeres forming the blastoderm (Fig. 1C). of the body wall (periblast) over the yolk sac The germ ring was visualized; then, epiboly had begun; the eyes were fully pigmented; an began. Gastration had begun and formation of increase in the number of melanophores had the embryonic shield was visible by 6 hours occurred, especially on the cephalic region and and 45 minutes after the first observation (Fig. lateral side of the body; the posterior tip of the Morphological Development of Mozambique Tilapia 297 Fig. 1. Egg development of Oreochromis mossambicus. (A) Blastocyst stage, 1 h 45 min, from the initial observation. (B) Morula stage, 3 h 30 min. (C) Blastula stage, 4 h 10 min. (D) Gastrula stage, 6 h 45 min. (E) Occurrence of the embryo, 22 h 27 min. (F) Nine somite stage, 26 h 17 min. (G) Appearance of the optic vesicle, 28 h. (H) Appearance of the auditory vesicle, 48 h 15 min. (I) Formation of the rudimental brain, 56 h 10 min. (J) Just before hatching, 75 h 15 min. notochord bent gradually upward; and buds of complete (Fig. 2E). At five days of age, the SL caudal fin rays had began to form (Fig. 2C). At was 6.0 mm, the number of melanophores had three days of age, the following was noted: the slightly increased, and the pectoral fin rays had NL was 5.7 mm; nostrils had formed; the body formed. wall continued to descend; and blood circula- At six days of age, the following was noted: tion was observed (veins had appeared in the SL was 6.6 mm; the free embryo was first swim- anterior portion of the yolk sac (Fig. 2D). At ming; and the buds of dorsal and anal fin rays four days of age, the standard length (SL) was had appeared (Fig. 3A). At seven days of age, 5.8 mm, and the caudal fin support was almost SL was 6.7 mm, guanine was present in the 298 K. Tachihara and E. Obara Fig. 2. Early development of Oreochromis mossambicus. (A) Newly hatched embryo, 4.0 mm in notochord length (NL). (B) One day old, 4.5 mm NL. (C) Two days old, 5.6 mm NL. (D) Three days old, 5.7 mm NL. (E) Four days old, 5.8 mm in standard length (SL). (F) Five days old, 6.0 mm SL. eyes, and the first segmentation of the soft SL was 9.4 mm; the visceral cavity was enclosed; rays of the dorsal fin had occurred (Fig. 3B). and formation of all the fins was complete, At eight days of age, SL was 7.1 mm, and mela- including the fin rays (Fig. 3E) The smallest nophores on the lateral side of the body had juvenile, which was collected in natural waters, increased density when compared to the previ- was 8.1 mm SL.