Proc. Indian Acad. Sci. (Anlm. Sci.), Vol. 90, Number 4, July 1981, pp. 417-426. © Printed in .

Embryonic and larval development of rohu-mrigal hybrid"

Y BASAVARAJU and T J VARGHESE University of Agricultural Sciences, College of Fisheries, Mangalore 575002, India

MS received 9 October 1980; revised 13 April 1981

Abstract. A detailed study of the embryonic and larval development of the major hybrid, rohu-mrigal, has been made and compared with the parents. The development of the hybrid was normal and more or less similar to that of the parental species, with only a few minor differences. The hybrid larvae exhibited intermediate range between the parental species in morphological features, such as nature of lips, presence of barbels and the shape of the caudal spot.

Keywords. Development; rohu-mrigal hybrid.

1. Introduction Hybridisation as a tool for improving natural stocks of plants and has been successfully applied to produce high yielding varieties in agriculture and husbandry. But its application in aquaculture is relatively a recent inno­ vation. After the development of the technique of hypophysation, a number of carp hybrids have been produced in India (Chaudhuri 1959, 1973; Varghese and Sukumaran 1971; Naseem Hamsa 1971; Naseem Hamsa and Alikunhi 1971; Konda Reddy 1977; Varghese and Shantaram 1979). But detailed evaluation of these hybrids from aquaculture point of view has been done only in a few cases. Therefore, a detailed study in development, taxonomic characters and growth of two major carp hybrids, rohu-rnrigal and mrigal-rohu, was taken up at the College of Fisheries, Mangslore. The present account deals with the embryonic and larval development of the hybrid, rohu-rnrigal,

2. Materials and methods The hybrid, rohu-mrigal was produced by crossing male rohu ( rohita) with female mrigal (Cirrhinus mrigala) employing the. technique of hypophysation. The method of dry stripping was followed. Wh12n the injected female mrigal was found freely oozing, the eggs were stripped into a clean enamel tray. Immediately

'" Formed part of the M.F.Sc. Thesis submitted by the senior author to the University of Agricultural Sciences, Bangalore, 417 418 Y Basavaraju and T J Varghese after this, male rohu was taken out and the milt was pressed on to the eggs. The sex products were then mixed thoroughly by gentle shaking of the tray. Excess of milt and slime were removed after a coupl of minutes by repeated washing of eggs with pond water. The fertilized eggs were kept in basins with sufficient water for water hardening and development. Following the same technique pure breeds of rohu and mrigal were also produced. Developing eggs of all the three breeds were transferred to separate hatching hapas, tied in cement cisterns, with appro­ priate labels. Before that, a sample of developing eggs were brought to the labo­ ratory for studying the development. The hatchlings, after yolk-sac absorption, were transferred to well-prepared nurseries. Samples from these were used for subsequent observations. The eggs were observed under a light source binocular microscope of 3·2 x 5 and 3·2 x 10 magnifications.. The observations were continuous from the time of fertilization till the completion of segmentation and thereafter at 30 min intervals till hatching. Mter hatching, observations were made at 6, 12, 24, 48 and 72 hr. Post-larval development was studied at intervals of 24 hr till the 7th day and thereafter on 10th, 12th, 15th, 20th and 25th days. For this, specimens freshly preserved in 4% formalin were used. Camera-lucida drawings were made under a magnification of 3·2 x 5 till the 7th day and thereafter, using 5 x or 10 x magnifications. The specimens were, however observed under higher magnifica­ tions, whenever necessary to study minute details. The developmental details of the rohu-mrigal hybrid only are described in this paper as these details of rohu and mrigal have already been published by Chakrabarty and Murty (1972).

Table 1. Embryonic development of rohu-mrigal hybrid

Range of water temperature: 26'0 to 30° c.

Hours after Progress in embryonic development fertilization

00·40 Blastodisc formation 00·45 2-celled stage (first cleavage) 00·50

s *4_ Development of rohu-mrigal hybrid 419 3. Results

3 .1. Embryonic development

The fully swollen eggs measured between 4·25 and 5·31 mm in diameter, with an average of 4·83 mm. The details of embryonic development of the hybrid are presented in table 1 and figure l(a) to (m).

3 .2. Larval development (figure 2a)

The just hatched larva is transparent and devoid of mouth. Eyes are pigmentless, The yolk-sac has two portions, anterior short bulbous portion and posterior elongated portion. Myotomes, 27 + 10. Total length 4·20 mm.

6 hr old hatchling (figure 2b)

The constriction between the bulbous portion and the elongated portion of the yolk-sac disappears. Two auditory concretions are clearly seen, the posterior one being slightly larger. Total length 4·44 mm.

12 hr old hatchling (figure 2c)

Eyes slightly pigmented at the centre. The bulbous portion of yolk-sac is reduced in height. Myotome.j Z? + 14. Pectoral fin bud appears. Notochord is seen as a spotted line. Total length 4·82 mm.

24 hr old hatchling (figure 2d)

Eyes become more pigmented, particularly at the centre. Mouth appears as a slit. Yolk-sac gets elongated. A reddish hue is seen near the opercular region. Total length 5'11 mm,

48 hr old hatchling(figure 2e)

Mouth opens. Air-bladder is seen. Pectoral fin is more prominent. Intestine is visible. Gill arches are seen as lines. Ventral fin-fold starts from about the middle region of air-bladder. Dorsal fin-fold starts immediately after the air­ bladder. Caudal is nearly round with five striations. A few chromatophores appear on the head. Total length 5·44 mm.

72 hr old hatchling (figure 2f) Yolk-sac almost completely absorbed. Mouth well-developed. Number of chromatophores on the head region increases. Operculum appears covering the gill-arches. Air-bladder elliptical in shape. Notochord clearly seen with the posterior end slightly upturned. Eight striations are present on the caudal fin. A few chromatophores are seen around the tip of the notochord, Myotomes, 27 + 14. Total len~h 5· 77llUll. 420 Y Basavaraju and T J Varghese

a b c Imm

d e f

9

J k .I -'"''

m

Figure 1. Embryonic development of rohu-mrigal hybrid. Fertilized egg­ (a) blastodisc formation, (b) 2-celJed stage, (c) 4-celJed stage, (d) 8-celled stage, (e) 16-celJed stage, (f) morula stage, (g) half yolk invasion, (h) yolk-plug stage, Embryo, (i) elongation of yolk mass, (j) comma stage without somites, (k) appear­ ance of optic cups, (1) appearance of Kupffer's vesicle, (m) embryo before hatching. Development of rohu-mrigal hybrid 421

L. hnm ...

hun

Figure 2. Pro-larval development of rohu-mrigal hybrid. Pro-larvae (a) just hatched, (b) 6 hr after hatching, (c) 12 hr after hatching, (d) 24 hr after hatching, (e) 48 lir after hatching, (0 72 hr after hatching.

3.3. Post-larval development

4th day after hatching (figure 30) Lips well-developed, the lower one being thicker and slightly shorter. Ventral fin-fold originates from about 3/4th the length of the air-bladder from the anterior end. Dorsal fin fold starts a little behind the air bladder. No constriction is seen in the air-bladder. Total length 6 mID, 422 Y Basavaraju and T J Varghese

(gl20lhday aftel: hatching,(hl2Sth .day after hatching.

Figure 3. Post-larval development of rohu-mrigal hybrid,. Post-larvae-(a) 4th day after hatching, (b) 5th day after hatching, (c) 6th day after hatching, (d) 7th day after hatching, (e) 10th day after hatching, (f) 15th day after hatching. ~) 20th day after hatching, (h) 25th day after hatching, Development of rohu-mrigal hybrid 423 5th day after hatching (figure 3b) The upper jaw is slightly longer than the lower jaw. Differentiation of dorsal fin has started, two pigmented striations appear. A small patch of dark pigments is seen below the tip of the notochord. Caudal with 12 rays, three rays on either side being unbranched. A small notch appears on the posterior margin of the caudal, indicating the commencement of bifurcation. Total length 6·55 mm.

6th day after hatching (figure 3c)

Pigmentation has increased on the body and head. Air bladder is covered by chromatophores, Dorsal fin with six striations. Dorsal fin fold still persists. Anal, separated from the ventra! fin-fold, no pigmentation seen. Caudal with 16 rays. Fresh fry looks more yellow-golden on the anterio-ventral side, while it is pale-yellow on the abdominal region. Total length 6·74 mm. 7th day after hatching (figure 3d) A constriction in the air-bladder appears resulting in an anterior globular part and a posterior elongated part. Ventral fin-bud appears. Dorsal fin is separated from the fin-fold, with eight slightly branched rays. Upper and lower jaws are more or less equal in length. Tip of the notochord clearly upturned. Caudal fork is marc clear, the lower lobe being slightly longer. Caudal with 18 rays, 4 on either side not branched. Total length 6 ·96 mm. 10th day after hatching (figure 3e)

Mouth well-developed; lips thick. A pair of short maxillary barbels can be seen under higher magnification. Gills are clearly visible. Ventral fin with two to three striations, the fin-fold still persists. Anal fin is not clearly differentiated and originates immediately after anal opening, being very close to caudal. The patch of chromatophores on the caudal peduncle region assumes a triangular shape. Dorsal with 2/13 rays. Caudal with 20 rays. Total length 10·37 mm. 12th day after hatching Lips are not yet fringed. Body pigmentation, particularly in the snout region, increases. Anal has 2/6 rays. Ventral with 6 rays, all unbranched. Base of the caudal is golden-yellow in colour.

15th day after hatching (figure 31) Lower lip is thicker than the upper one and slightly fringed, which is visible only under higher magnification. Barbels are still minute. Dorsal with 16 rays. Caudal fin with 22 rays. The triangular patch of chromatophores on the caudal peduncle becomes darker, Snout, pointed. Total length 16·61 mm. 'JiJth day after hatching (figure 3g) Maxillary barbels become quite clear and visible to naked eyes. Body almost completely covered with pigments, more so on the head and on the dorsal side above the lateral line. Lower lip is thick! and fringed. Caudal, deeply forkQd. 424 Y Basavaraju and T J Varghese

Triangular band in the caudal peduncle region is more prominent. A few scales appear on the region posterior to the operculum. Total length 20·88 mm. 25th day after hatching (figure 3h) Scales are well formed, covering almost the entire body and are dark on their margin. Mouth, inferior. Lower lip fringed. Body fully pigmented. Triangular band on the caudal peduncle is very prominent. Dorsal, 3/13 with pigmentation at the base. Anal, 2/6; ventral with 9 rays. Caudal deeply forked with 26 rays. Fry, when fresh, appear light-green along the dorsal region. Total length 23·35 mm.

4. DiscassioD During the present study, it was observed that the development of the hybrid, rohu-mrigal was normal and more or less similar to that of the parental species, with a few minor variations. The size of the fully swollen eggs of the hybrid was intermediate between those of the parental species. Konda Reddy (1977) reported that fully swollen eggs of -rohu hybrid were bigger than those of the parents, while they were smaller in case of the reciprocal hybrid, rohu-catla. Thus, it can be concluded that the size of eggs is not of much significance in demarcating carp hybrids and their parental species. The variation in size of developing carp eggs may be due to variation in water temperature (Chakrabarty and Murty 1972).

4 .1. Embryonic development The rate of development of fish embryo varies according to the fluctuation of water temperature. Chakrabarty and Murty (1972) noted the commencement of first clevage in major 45 min after fertilization. Komia Reddy (1977) stated that the commencement of clevage in catla-rohu and rohu-catIa hybrids was inter­ mediate between that of parental species (49 min in catla, 33 min in rohu, 45 min in catla-rohu and 35 min in rohu-catla). But the results of the present study are not in agreem nt with the above findings as the clevage in rohu-mrigal hybrid started later than the parental species (40 min in mrigal, 33 min in rohu and 45 min in the hybrid). This indicates that the time taken for first clevage in major carps and their hybrids is subjected to great variation. Incubation period of eggs of major carps and their hybrids, range from 14 to 18 hr (Mookerjee 1945; Chakrabarty and Murty 1972; Konda Reddy 1977). In the present study, it was noticed that the incubation period was less than that of both the parental species. Konda Reddy (977) found that the incubation period in the hybrids, catla-rohu and rohu-catla to be intermediate between the parental species.

4 .2. Larval development As observed by Chakrabarty and Murty (1972), in case of major carps and ,by Konda Reddy (1977) in case of hybrids between carla and rohu, the newly hatched larvae in the present study also were transparent, pigmentless and devoid,of mouth and gills. The pigmentation was seen for the first time in the 12 hr old hatchlingsas reported by earlier workers. The pectoral fin-bud also appeared: Development of rohu-mrigal hybrid 425 in the 12 hr old hatchlings, in case of major carps and their hybrids. In the present investigation, though the intestine was formed two days after hatching in all the three breeds, no food particles wei e noticed till the third day, whereas Konda Reddy (1977) reported that the intestine of the 2-day old lai vae of catla­ rohu and rohu-catla hybrids had algae and some cladocerans inside.

4.3. Post-larval development The yolk-sac was completely absorbed in the hybrid and parental species on the 4th day. Similar observations have been recorded by Chakrabarty and Murty (1972) in rohu and mrigal and in respect of catla, rohu and their hybrids by Konda Reddy (1977). The dorsal fin rudiment appeared on the 5th day in the hybrid, while in parents it appeared only on the sixth day. In 6-day old hatchling of the hybrid, the dorsal fin was completely separated. Konda Reddy (1977) reported the appearance of dorsal fin with 5 rays on the Sth day in the catla-rohu hybrid, but there were no rays in the reciprocal hybrid of same age. As observed by earlier workers (Chakrabarty and Murty 1972; Konda Reddy 1977), all the three breeds presently studied had a constricted air-bladder on the 6th day. However, the constriction was completed only on the 7th day. According to Chakrabarty and Murty (1972) and Konda Reddy (1977) both anal fin and pelvic fin-bud appeared on the 6th day, whereas in the present study, the anal fin appeared on the 6th day and the pelvic fin-bud on the 7th day. A pair of short maxillary barbels appeared on the 12th day in rohu and on the 10th day in rohu-mrigal, whereas the barbels were absent in mrigal even on 25th day. Similar observations were made by Chakrabarty and Murty (1972) in respect of rohu and mrigal. Konda Reddy (1977) observed the appearance of a pair of maxillary barbels on the 10th day in rohu, catla-rohu and rohu-catla. In respect of catla-rohu and rohu-catla hybrids, Konda Reddy (1977) reported that only the lower lip was fringed, displaying an intermediate character between the parental species. In the present study also, in the hybrid rohu-mrigal, only the lower lip was fringed and it was noticed on the 15th day. In case of rohu the lower lip was found to be fringed on the 10th day itself as reported by Chakrabarty and Murty (1972). During the present study the scales appeared on the body for the first time on the 20th day in all the three breeds and the entire body was covered by scales on the 25th day. However, Konda Reddy (1977) observed that in both the hybrids, catla-rohu and rohu-catla, scales appeared on the 15th day itself and the entire body was covered with scales on the 20th day. The caudal spot was triangular in the rohu-mrigal hybrid, indicating an intermediate character between the parental species, since it is band shaped in rohu and diamond shaped in mrigal. Konda Reddy (1977) also reported that the caudal spot in case of the hybrids, catla-rohu and rohu-cetla, was intermediate between parental species. From the data pre­ sented herein it can be concluded that the development of rohu-mrigal hybrid was normal, similar to that of the parental species. The hybrid exhibited inter­ mediate characters between the parental species in most of the morphological features (Basavaraju and Varghese 1980). 426 Y Basavaraju and T J Varghese Acknowledgement The authors express their sincere thanks to Prof. H P C Shetty, Director of Instruction, College of Fisheries, Mangalore, for his constant encouragement during the course of this investigation. The ICAR junior fellowship awarded to the first author is gratefully acknowledged.

References

Basavaraju Y and Varghese T J 1980 The taxonomic characters of rohu-mrigal and mrigal-rohu hybrids; Mysore J. Agric. Sci. 14 567-573 Chakrabarty R D and Murty DS 1972 Life history of Indian major carps, Cirrhinus mrigala (Ham.), Catla catla (Ham.) and Labeo rohita (Ham.); J. Inland Fish. Soc. India 4 132-161 Chaudhuri H 1959 Experiments on hybridisation of Indian carps; Proc. Indian Sci. Congr, (Abstr.) 46 20-21 Chaudhuri H 1973 Fertility of th.e hybrids of Indian carps and preliminary studies on the F, generation of carp hybrids; J. Inland Fish. Soc. India 5 195-200 Konda Reddy P 1977 Studies on the development, taxonomic characters and growth of catla-rohu and rohu-catla hybrids; M.F.Sc. Thesis, Univ. Agril. Sci., Bangalore p. 136 Mookerjee HK 1945 Life history of some major carps of Bengal; Sci. Cult. 10 400-402 Naseem Hamsa K 1971 Preliminary account of an intergeneric fertile hybrid between Cirrhina mrigala and Labeo rohita; Annual day souvenir C.I.F.E. Bombay pp. 10-15 Naseem Hamsa K and Alikunhi K H 1971 Preliminary studies on • upgrading' Cirrhina reba by hybridisation ; J. Indian Fish. Assoc. 1 68-73 Varghese T J and Sukurnaran KK 1971 Notes on hypophysation of catla x rohu hybrid; Annual Day Souvenir, C.I.F.E., Bombay pp. 16-17 Varghese T J and Shantaram B 1979 Preliminary studies on the growth rates of three Indian major carp hybrids; Proc. Indian Acad. Sci. (Anim. Sci.) 88 209-216