Development and Species Identification of the Ricefish (Genus Oryzias) in Thailand

Apichart Termvidchakorn 1 and Wichian Magtoon 2 1 Prachinburi Inland Fisheries Station. Inland Fisheries Research and Development Bureau, Department of

Fisheries. Krabinburi, Prachinburi Province 20500 Thailand. 2 Faculty of Science, Srinakharinwirot University at Prasarnmitra.

Abstract There are 4 species of the genus Oryzias in Thailand. These 4 species are O. javanicus and O. dancena which occurr in brackish water and coastal area of southern part of Thailand, O. mekongensis distributed in north eastern part of Thailand and Mekong River Basin, O. minutillus has been reported in all part of Thailand. Larvae of these 4 species were studied about their meristic characters and chromatophore pigment development pattern. The represent of 3 stages ie. Larval , post larval and juvenile were used for study about their species identification. The chromatophore pigment pattern, number of myomere, dorsal and anal fin ray were used as the meristic characters for identification. Introduction Ricefish of Medaka of the genus Oryzias Jordan and Snyder was small fish distributed from freshwater to brackish water. There were 4 species of ricefish in Thailand (Magtoon et al. 1992). The Thai Medaka, O. minutillus (Smith, 1945) was a small fish commonly in pond. Ditch and paddy fields in the central, north, northeast and southern part of Thailand and also reported from Xishiangbanna, Yunan China and Rangoon, Myanmar (Uwa et al., 1988). Another small species of O. mekongensis Uwa and Magtoon 1986 were found at the northeastern part of Thailand and Mekong river basin area. For the brackish water species of O. dancena (Hamiton, 1822) and O. javanicus (Bleeker, 1848) were found in the southern part of Thailand. While the O. dancenawas wild distribution from India, Sri Lanka and Myanmar. The O. javanicus also distributed from southern part of Thailand to Malaysia, Singapore and Indonesia. The ricefish had a general description as : Body laterally compressed, head depressed, eye moderately large, the number of vertebrate were 28-31, dorsal fin base situated from 17th to 20-21st vertebrae spine and the anal fin base situated from 10-11st to 19-20th vertebrae spine. The number of dorsal and anal fin rays were differented among species. O. javanicus were 6(6-7) for the dorsal fin ray and 23(20-23) for the anal fin rays. The number of dorsal and anal fin rays of O. dancenawere 7 and 24(22-24) while O. minutillus were 6(5-7) and 19(18-21) for the number of dorsal and anal fin rays count. O. mekongensis were 6(5-7) and 14(13-16), respectively. Iwamatsu (1994) studied the developmental stages of the medaka (O. latipes) from unfertilized eggs, fertilized eggs to hatching stages of embryo and from the hatching larvae to young stage of medaka. The just hatched larvae of medaka had further development which showed from the development of eye, mouth part and caudal fin. The number of fin rays and chromatophore pigment pattern were used for species identification of larvae .

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Material and Method The specimens were collected from breeding tank at the different age from hatching to juvenile stage. The larvae had divided to just hatch larvae (larval stages) , post larval stage which using the stage that pelvic fin was started to developed, and juvenile stage started when all of the meristic characters were completely developed and scale were start to developed. 5 specimens of each stage were collected and preserved with 10% formalin solution and change to 4% formalin solution after 2 weeks. The developmental stages and identification were studied at the laboratory under the stereomicroscope and drawing with camera lucida for each stage. The different among meristic characters and chromatophore pigment pattern were studied for their species identification. The stages of the ricefish larvae were: Larval stage : This stage started from the larvae hatched out of the egg shale and ended at the stages that the pelvic fin were started to develop. Post larval stage : This stage started from the pelvic fin was forming and finished when the scale were started to develop. Juvenile stage : This stage started at the time that the scale were started to develop and finished when the scales were completely developed.

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RESULTS 1. The development of the ricefish larvae. Ricefish of the genus Oryzias showed a long embryonic development in their incubation period. When the larvae hatched out from the egg shell; some part of their body had developed pass through the yolk sac stage and showed the larval period characters. 1.1 The developmental stages of the southern ricefish (O. javanicus) collected from the rearing pond. The 4.10 millimeter of the just hatch larvae (Fig. 2 A). The body of the larvae was large and laterally compressed. The head was slightly depress, mouth was opened, maxilla and mandible were developed, large eye about one third of the head. The gut opening was about one third of the body a head. Urostyle was flexion and the support bone (pleural and hyplural bone) were developed. The pectoral, dorsal and anal fanfold were present. The caudal fin rays were started to develop. The chromatophore pigment were present on head, operculum, mid line dorsal and ventral part of the body. The 5.37 millimeter of 1 day old larvae (Fig. 2 B). The development of this stage were the mouth part which were more practical. The support caudal bone and rays were developed. The chromatophore pigment was increase on their head, mouth, dorsal, ventral mid line of the body and on the caudal ray. The 6.47 millimeter of 3 day old larvae (Fig. 2 C). The charge of this stage were the dorsal and anal fins were started to develop from the anterior part of the dorsal and anal fin fold. The chromatophore pigment was increasing on head, cheek and anterior part of the gut. The 7.75 millimeter of 5 day old larvae (Fig. 2 D). The development of these stage were the formation of dorsal, anal and pectoral fin rays after the fin folds had been developed their shape to these fins. The fin fold were also decreased their size. The chromatophore pigment was increasing on head gut and caudal fin rays. The 9.67 millimeter of 7 day old larvae (Fig. 3 A). These stage showed the development of the dorsal, anal and pectoral fin rays while the pelvic fin were started to form at the ventral part of the body in front of the anus. The chromatophore pigment was increased on head, operculum, mandible gut, dorsal part of the body and caudal fin rays. The 12.94 millimeter of 9 day old larvae (Fig. 3 B). The larvae had a carpet development of dorsal, anal, pectoral and caudal fin rays while the pelvic fin had more developed on size and rays. The chromatophore pigment were increasing on head, operculum, mandible gut, dorsal part of body. The 15.88 millimeter of 12 day old larvae (Fig. 3 C). The larvae had complete development on their fin rays 6 dorsal rays, 23 anal rays, 10 pectoral rays and 6 pelvic rays. The chromatophore pigment were increased on dorsal part of body above mid body line. The 19.67 millimeter of 15 day old larvae (Fig. 3 D).The juvenile ricefish had completely developed on their meristic characters and increased the chromatophore pigment on their head and body. The scale was formed on the body and the chromatophore pigment were also present on the caudal rays.

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1.2 The developmental stages of the brackish water ricefish (O. melastigma). The 5.00 millimeter of 1 day old brackish water larvae (Fig. 4 A). The body was laterally compressed, head slightly depress, eye large, month opened and function. The gut opening was about one third of the body, urostyle was flexion and the supported caudal bone was developed. Pectoral fin was formed and base of the fin was above the mid line of the body. The caudal fin rays were developed. The dorsal and anal fin fold were present. Chromatophore pigment was present on dorsal part of head opercle anterior part of the gut, dorsal ventral and mid line of the body. The 6.64 millimeter of 3 day old brackish water larvae (Fig. 4 B). The dorsal and anal fins was developed from the anterior part of dorsal and anal fin folds. The caudal fin rays were formed. Chromatophore pigment was increased on their head operculum maxilla and mandible of the mouth part, anterior part of the gut, dorsal ventral and mid line of the body. The 7.64 millimeter of 5 day old brackish water larvae (Fig. 4 C). The dorsal and anal fins fold had formed the dorsal and anal fins. The dorsal anal and pectoral fin rays were developed. The chromatophore pigment occurred on pectoral fin and increased on their head, operculum mouth part, gut and on the body. The 9.53 millimeter of 7 day old brackish water larvae (Fig. 4 D). The larvae was elongate to post larvae stage by showing the formation of the pelvic fin which was the last pair fin occurred at the ventral part of the body before the anus. The dorsal anal and pectoral fin rays were more developed. The chromatophore pigment were increase on gut, dorsal, ventral part of the body, on the pectoral and a longitudinal band occurred at the mid of the anal fin. The 13.07 millimeter of 9 day old brackish water larvae (Fig. 5 A). The anal fin rays were completely developed as their basic number of 23 rays. The dorsal and pectoral fin rays were developing. The pelvic fin was increased on size. The chromatophore pigment increase on head, dorsal and ventral part of the body. The 16.58 millimeter of 12 day old brackish water larvae (Fig. 5 B). The head of the larvae more point than before. The fin rays were completely developed as their basic number for dorsal fin was 7 rays and pectoral fin was 10 rays which the size of pelvic fin was increasing and the rays were started to develop. The chromatophore pigment were increased on head, body, pectoral and anal fin. The 20.48 millimeter of 15 day old brackish water larvae (Fig. 5 C). The inferior mouth was clearly showed in this stage all fin rays were completely developed. The chromatophore pigment was increasing on their head, body, pelvic and anal fin. The 24.35 millimeter of 19 day old brackish water larvae (Fig. 5 D). The fish showed completely development on meristic characters and the scales were forming at this stage. The reproduction organ was not developed yet. 1.3 The developmental stages of the Mekong ricefish (O. mekongensis). The 4.95 millimeter of 1 day old Mekong ricefish larvae (Fig. 6 A). The body was elongate and laterally compressed, head slightly depress, month was forms already. The eye diameters was more than one third of head length. The urostyle was flexion and the support caudal bone were formed. The caudal fin was developed from the fin fold at the caudal part. The pectoral, dorsal and anal fin fold were presented. The caudal rays were

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forming. The chromatophore pigment was presented on head snout anterior part of the gut, dorsal anal and mid line of the body and also on the caudal ray. The 6.63 millimeter of 1 day old Mekong ricefish larvae (Fig. 6 B). The larvae of these stage showed the formation of dorsal and anal fins from changing the shape of the fin fold. The chromatophore pigment was increasing on head, gut dorsal ventral and mid line of the body. The 7.33 millimeter of 5 day old Mekong ricefish larvae (Fig. 6 C). The larvae was started to from their pectoral, dorsal and anal rays. The chromatophore pigment was increasing head, operculum, maxilla, mandible and pectoral fin. The pigment on the caudal fin was changed their pattern from scattering to the strip at the upper and larvae rim of the caudal fin. The 9.53 millimeter of 7 day old Mekong ricefish larvae (Fig. 6 D). The larvae started to from the pelvic fin at the ventral part of body before the anus. The dorsal rays was completely developed while pectoral and anal rays were developing. The chromatophore pigment was increased on head and mandible infront of the eye. The 12.10 millimeter of 9 day old Mekong ricefish larvae (Fig. 7 A). The pectoral fin rays was completely developed while the size of pelvic fin was increased and ray was forming. The chromatophore pigment increased on head dorsal and ventral parts of the body. The 14.04 millimeter of 12 day old Mekong ricefish larvae (Fig. 7 B) The larvae in this stage showed the completely development of all fin rays. The fin ray number of dorsal fin, anal fin, pectoral fin and pelvic fin were 6, 14, 8 and 6 respectively. The chromatophore pigment occurred on dorsal fin and increased on head, caudal fin, dorsal and ventral parts of the body. The 18.25 millimeter of 15 day old Mekong ricefish larvae (Fig. 7 C). The chromatophore pigment increased on their body cover all myomeres. The chromatophore pigment strips on the caudal fin was clearly showed off. The 20.40 millimeter of 19 day old Mekong ricefish larvae. (Fig. 7 D) The larvae had completely developed on their meristic characters. The scales were developing at this stage and the larvae changed from post-larvae to juvenile when the scale was forming. 1.4 The developmental stages of the Thai ricefish (O. minutillus). The 5.28 millimeter of 1 day old Thai ricefish larvae (Fig. 8 A). The body was elongate and laterally compressed, head slightly depress. The superior month was small and the eye diameters was about one third of head length. The urostyle had already flexion and the caudal supported bone were formed. The pectoral, dorsal and anal fin fold were presented. The caudal fin was developed from the fin fold at the caudal part and the caudal ray were forming. The chromatophore pigment was presented on head, operculum, pectoral fin base, dorsal, ventral and mid line of the body as a small datch. The 5.88 millimeter of 3 day old Thai ricefish larvae (Fig. 8 B). The larvae showed the formation of dorsal and anal fin from changing the shape of fin fold. The chromatophore pigment increased on head gut, dorsal, ventral and mid line of the body. The 8.09 millimeter of 5 day old Thai ricefish larvae (Fig. 8 C). The head became small and pointed. The pectoral and anal fin rays were started to develop while the dorsal ray

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was not developed yet. The chromatophore pigment were increase on dorsal anal and mid line of the body. The 9.57 millimeter of 7 day old Thai ricefish larvae (Fig. 8 D). The shape of the dorsal fin was more clear than the previous stage and the pelvic fin was forming as a small double fin fold at the ventral part of the body infront of the anus. The pectoral and anal rays were developing. The chromatophore pigment increased on their head and mandible. The 11.13 millimeter of 9 day old Thai ricefish larvae (Fig. 9 A). The larvae had developed on their fin rays. The size of pelvic fin was increasing while the fin fold between gut and anus was absent. The chromatophore pigment were increasing on doro - leteral part of the body. The 12.82 millimeter of 12 day old Thai ricefish larvae (Fig. 9 B). The larvae in this stage. the pectoral and dorsal fin rays were completely developed. The anal fin rays was developing and the pelvic fin was started to form. The chromatophore pigment occurred on head mandible doro - leteral and mid line of the body. The 16.15 millimeter of 15 day old Thai ricefish larvae (Fig. 9 C). The rays of all fin had completely developed. The number of the pectoral fin, dorsal fin, anal fin and pectoral fin were 8, 6, 18 and 5 respectively. The chromatophore pigment occurred on head dorsal ventral and mid line of the body. The 19.86 millimeter of 19 day old Thai ricefish larvae (Fig. 9 D). The larvae had changed their stage from post larvae to juvenile, that observed from the formation of the scale. The chromatophore pigment was increased on head, dorsal and ventral part of the body. The scale was formed the skin where the pigment was dense. 2. Key to identified genus Oryzias. The larvae of the genus Oryzias was identified to species by using the difference of mophomatics and chromatophore pigment pattern in each stage. 2.1 Key to identified the larval stage of Oryzias larvae. (Fig 10 A – D) 1a Chromatophore pigment present on caudal fin…………..…………………………….. 2 1b Chromatophore pigment absent from caudal fin ……….……………...……………… 3 2a Chromatophore pigment present on base of pectoral fin and pre anal myomere …..………………………………………………..O. javanicus 2b Chromatophore pigment absent from been of pectoral fin and preanal myomere …..……………………………………………..O. mekongensis 3a Chromatophore pigment present on base of pectoral fin and pre anal myomere …..………………………………………..…………..O. minutillus 3b Chromatophore pigment absent on base of pectoral fin and pre anal myomere …..………………………………………...………….. O. dancena 2.2 Key to identified the post larval stage of Oryzias larvae. (Fig 11 A – D) 1a Chromatophore pigment present on caudal fin…………..…………………………….. 2 1b Chromatophore pigment absent from caudal fin ……….……………...……………… 3 2a Heavy pigment on gut and body……………...…………………………..O. javanicus

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2b Few pigment on gut and body……………………………………..…..O. mekongensis 3a Heavy pigment on gut and body……………...……………………..………..O. minutillus 3b Few pigment on gut and body……………………………………………..….. O. dancena 2.3 Key to identified the juvenile stage of Oryzias larvae. (Fig 12 A – D) 1a Chromatophore pigment present on caudal fin…………..…………………………….. 2 1b Chromatophore pigment absent from caudal fin ……….……………...……………… 3 2a Chromatophore pigment scatter on caudal fin and absent on dorsal fin. Anal fin ray 23..………….…………………….….. O. javanicus 2b Chromatophore pigment showed strip band on dorsal and ventral part of caudal fin and dorsal fin. Anal fin ray 14……….....… O. mekongensis 3a Chromatophore pigment present on anal fin . Anal fin ray 24……..………..O. minutillus 3b Chromatophore pigment absent on anal fin . Anal fin ray 18………………... O. dancena Discussion There were 4 species of the fish in genus Oryzias which occurred in Thailand (Magtoon and Uwa, 1988). O. javanicus and O. dancena distributed in brackish water and costal area, O. mekongensis were found in north eastern part of Thailand or along Mekong river (Uwa and Magtoon, 1986) while O. minutillus were found in all part of Thailand (Smith, 1945). The Oryzias larvae was a well development on the head, eye, mouth, digestive tract and caudal part. When it hatch out, the larvae had pass the prelarval stage. The yolk had completely absorbed. At the caudal part, the flexion urostyle was showed while the supported bone were forming. The chromatophore pigment presented on their head, dorsal ventral and mid line of the body. The chromatophore pigment was increasing on their head, body and ray in each stage which was the same as the studied of Iwamatsu (1994) in the stage of normal development of Medaka (Oryzias latipes). The identification on their meristic character ie. Number of dorsal anal pectoral and pelvic rats and complied with the chromatophore pigment pattern on their head body and rays. Reference Magtoon W. and Uwa, H. 1988. First Report of Oryzias melastigma (Cyprinodontidae:

Pisees) in Thailand. 26th Kasatsart University Sciantific Symposium. P. 229-233. Magtoon W. 1987 (a). Distribution and Morphological study of the fish in genus Oryzias in

southern part of Thailand. Report of the National Research … of Thailand. 13 p. Magtoon W. 1987 (b). Study on some biological aspect of the O. minutillus in central and

northern part of Thailand. Report of Srinakharinwirot University. 23 p. Smith, H.M. 1945. The Fresh-Water Fishes of Siam or Thailand. Bull. USA. Nat. Mus. 188 :

1-622. Uwa, H. and Magtoon W. 1986. Description and Karyotype of a new ricefish, O.

mekongensis, from Thailand. Copeia, 1986 (2) 473-477. Magtoon W., N. Nadee, T. Higastitani, K. Takata and H. Uwa. 1922. Karyotype evolution

and geographical distribution of the Thai – medaka, O. minutillus, in Thailand.. Journal of Fish Biology (1992) 41: 489-497.

Iwamatsu, T. 1994

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Figure 1 Meristic character of the Ricefish larvae. A. larval stage B. post larval stage and C. juvenile.

Total Length

Notochord Length Dorsal fin fold Urostyle

Anus Operculum

Eye

Anal fin fold

Myosepta

Myomere

Snout Pectoral fin

Dorsal fin

Caudal fin

Pelvic fin Anal fin

Total Length

Standard Length

Pre-dorsal Length Pre-anal Length

Head Length

A.

B.

C.

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Figure 2 The developmental stage of the Southern Ricefish (O. javanicus (Bleeker, 1848)) from artificial breeding. A. 4.06 mm.of the just hatch larval.B. 5.37 mm.of 1 day old larvae. C. 6.47 mm.of 1 day old larvae and D. 7.75 mm.of 5 day old larvae.

A.

1 mm.

1 mm.

B.

C.

D.

2 mm.

2 mm.

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Figure 3 The developmental stage of the Southern Ricefish (O. javanicus (Bleeker, 1848)) from artificial breeding. A. 9.67 mm. of 7 day old larvae. B. 12.94 mm.of 9 day old larvae. C. 15.88 mm.of 12 day old larvae and D. 19.67 mm.of 15 day old juvenile.

A. 3 mm.

3 mm.

B.

C.

D.

3 mm.

3 mm.

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Figure 4 The developmental stage of the Brackish Water Ricefish (O. dancena (Hamilton, 1822)) from artificial breeding. A. 5.00 mm.of 1 day old larvae. B. 6.64 mm.of 3 day old larvae. C. 7.64 mm.of 5 day old larvae and D. 9.53 mm.of 7 day old larvae.

A. 1 mm.

1 mm. B.

C.

D.

2 mm.

2 mm.

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Figure 5 The developmental stage of the Brackish Water Ricefish (O. dancena (Hamilton, 1822)) from artificial breeding. A. 13.07 mm.of 9 day old larvae. B. 16.58 mm.of 12 day old larvae. C. 20.48 mm.of 15 day old larvae and D. 24.35 mm.of 19 day old juvenile.

5 mm.

A.

3 mm.

3 mm.

B.

C.

D.

5 mm.

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Figure 6 The developmental stage of the Mekong Ricefish (O. mekongensis Uwa and Magtoon, 1986) from artificial breeding. A. 4.95 mm.of 1 day old larvae. B. 6.63 mm.of 3 day old larvae. C. 7.33 mm.of 5 day old larvae and D. 9.53 mm.of 7 day old larvae.

2 mm.

A. 1 mm.

1 mm.

B.

C.

D.

2 mm.

3 mm.

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Figure 7 The developmental stage of the Mekong Ricefish (O. mekongensis Uwa and Magtoon, 1986) from artificial breeding. A. 12.10 mm.of 9 day old larvae. B. 14.04 mm.of 12 day old larvae. C. 18.25 mm.of 15 day old larvae and D. 20.40 mm.of 19 day old juvenile.

3 mm.

A. 3 mm.

3 mm.

B.

C.

D.

3 mm.

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Figure 8 The developmental stage of the Thai Ricefish (O. minutillus Smith, 1945) from artificial breeding. A. 5.28 mm.of 1 day old larvae. B. 5.88 mm.of 3 day old larvae. C. 8.09 mm.of 5 day old larvae and D. 9.57 mm.of 7 day old larvae.

2 mm.

A.

1 mm.

1 mm. B.

C.

D.

2 mm.

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Figure 9 The developmental stage of the Thai Ricefish (O. minutillus Smith, 1945) from artificial breeding. A. 11.13 mm.of 9 day old larvae. B. 12.82 mm.of 12 day old larvae. C. 16.15 mm.of 15 day old larvae and D. 19.86 mm.of 19 day old juvenile.

3 mm.

A. 3 mm.

3 mm.

B.

C.

D.

3 mm.

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Figure 10 Showed the difference of the meristic characters and chromatophore pigment

pattern among the species of larval stage of Ricefish. A. 5.37 mm.of Oryzias javanicus B. 5.00 mm.of Oryzias dancena C. 4.95 mm.of Oryzias mekongensis and D. 5.28 mm. of Oryzias minutillus

1 mm.

1 mm.

B.

C.

D.

1 mm.

1 mm.

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Figure 11 Showed the difference of the meristic characters and chromatophore pigment

pattern among the species of post larval stage of Ricefish. A. 9.67 mm.of Oryzias javanicus B. 9.53 mm.of Oryzias dancena C. 8.61 mm.of Oryzias mekongensis and D. 9.57 mm. of Oryzias minutillus

1 mm.

2 mm.

2 mm.

A. 2 mm.

B.

C.

D.

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Figure 12 Showed the difference of the meristic characters and chromatophore pigment

pattern among the species of juvenile stage of Ricefish. A. 19.67 mm.of Oryzias javanicus B. 24.35 mm.of Oryzias dancena C. 20.40 mm.of Oryzias mekongensis and D. 19.86 mm. of Oryzias minutillus

3 mm.

A.

3 mm.

5 mm.

B.

C.

D.

3 mm.

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