Embryonic and larval development of the striped mullet Mugil eephalus CL)

MATHEW ABRAHAM, P. SHIRANEE, P. KISHORE CHANDRA, M. KAILASAM AND V.K. CHARLES

Central Institute of Brackishwater Aquaculture, Madras-600 008, India

ABSTRACT

Mugil cephalus in ripe condition collected from inshore catches at Muttilkadu near Madras were induced to breed using HCG and ovaprim to study the embryonic and larval development. Samples of unfertilised eggs and various developmental stages from fertilization through embryonic and larval stages were preserved for further micrographic studies. Rate of fertilization was determined at the time of blastodisc formation and was 90 %. Of the total number of 1.46 million eggs, 1.26 million were fertilised. The pre-hatched embryo was deeply pigmented, exhibited twitching movements and lay curved over the yolk mass. After an incubation period of 30-32 hrs, the embryo hatched out. The newly hatched out larvae measuring 2.29 mm, were transparent with a large oval head, a well defined yolk sac and short tail encircled by a continuous finfold. hlouth formation was complete and the larvae started feeding on 3rd day of hatching. The paper presents the results of detailed studies of embryonic and larva1 development and the time scale established for critical stages of development.

Introduction

Embryonic and larval development studies besides providing interesting information in itself, are imperative and consequential to the successful rearing of larvae for seed production. Despite the successes in artificial propagation of the mullet, by induced spawning, there is still a need to refine further the techniques of larval rearing particularly for practical and commercial applica- tions (Liao, 1993). It is an undisputed fact that larval rearing remains the most critical and crucial phase in brackishwater fish seed production. Development of suitable protocols for the mass rearing of larval fish repre-

sents one of the last bamers for the successful propagation of a variety of marine species (Tamaru et al., 1993). Most problems arise from relatively smaller size of the mouth and limited yolk reserves (Shirota, 1970) of the larvae.

The present study elaborates with photographic evidence the embryonic and larval structure and development with the corresponding time scale of M. cephalus. A photographic presentation of the embryonic development of M. cephalus with time scale has been elaborated by Tamaru et al. (1993). Vivid photographs, however, with no textual reference have been made by

Mathew Abraham e t al.

Liao (1993). Earlier studies on the development of the mullets, conducted on various geographic and environrnen-/ tal profiles include that of Nair (1957),: Anderson (1958), Kuo et al. (1973), Ling; (1970) and Chaudhuri et al. (1977). i Materials and methods i

Mugil cephalus breeders were ob- \

tained from commercial sea catches a t Muttukadu, near Madras during Febru- ary 1997. Females with an average oocyte diameter of >525pm and milting males were selected. The selected fish were given a dip treatment in 1 ppm acriflavin and 1 hr bath in 10 ppm oxytetracyclin. Induced breeding was done following the two injection protocol with a 24 hr interval. HCG was used as the priming dose @ 6,000-10,000 I.U./kg body weight and ovaprim was used as the resolving dose @ 3-5mIJkg body weight. The males were not given any hormone treatment. Dry stripping was resorted to and fertilization was effected in-vitro. Incubation was done @ 140 eggsA. Water temperature ranged from 26-28°C and salinity was 26 ppt. Sam- ples of eggs before fertilization, imme- diately after fertilization and there- after a t 30 minute intervals until hatch- ing and including the larval samples at 30 minute intervals were also preserved in a medium made of 2 % formalin, 4 % glycerol and 94 % water for further studies. The larvae were reared in FRP tanks provided with flow through run- ning water system at a density of 45-50 nosll. The larvae were fed with the rotifer Brachionus plicatilis from third day onwards.

Results

The ovulation was complete 22 hrs after the resolving dose. The stripped eggs were translucent and non adhesive

with a deep yellow colour. The diameter of the spawned eggs ranged from 750- 760 pm. The yolk was characterised by a single yolk globule and the eggs were telolecithal in nature (Plate 1 A). Fer- tilization was effected by stripping milt from males and mixing it with the eggs. The total number of eggs was estimated -&-?&1.4 m and the rate of fertiliz 'on 90 %. Incubation period spann / over a period of 30-32 hrs and the hatching rate was 41.6 %. Immediately aRer fertilization the germplasm migrated to the animal pole to form a dense cap like structure called the germinal disc (Plate 1B). Cleavage (Plate 1 C,D,E,F) was restricted to the germinal disc and the resulting blastoderm assumed a disc like multi-cellular structure called the blastodisc, which was more or less convex in shape and enclosed between itself and the uncleaved residue of the egg - a cavity representing the blasto- coele (Plate 1G) cleacage was followed by gastrulation which converted the embryo into a two layered structure (Plate I H). The outer germ layer, the epiblast, gave rise to the ectoderm while the involuted cell mass, the hypoblast, the endodermal and mesodermal com- ponents.

Subsequent to gastrulation, develop- ment continued until a primitive verte- brate body was formed and the neural plate established. The embryo was then more or less cylindrical and bilaterally symmetrical and was referred to as neurula (Plate 1. I). The head and pharyngeal region projected from the yolk mass anteriorly and the trunk curved over the yolk and the tail projected posteriorly (Plate 1. J). The mesoderm on both sides of the noto- chord organised into somites and melanophores began to appear (Plate 1 K). The pre-hatched embryo showed

Embryonzc and larval development of hlugil cephalus 125

Platc: 1. '4. Fertilised egg, B. Germinal disc stage, C. 2-cell stage, U. &cell stage, E. 16-cell stage, F. 32-cell stage, G. Morula stage, H. Gastrula, I. Early neurula, J. Neurula somite stage, K. Late neurula, L. Pre-hatched embryo. (Magnification: x 40).

Embryon~c and larual development of Mug1 cephalus 127

Plate 2. A. Newly hatched larva, B. 24-hr old larva. (Magnification: x 100).

Mathew Abraham e t al.

length of 2.80 mm and 60 % of the yolk was utilised. The mouth opening meas- ured 135 pm in the 96 hrs old larvae (Plate 3 B). The alimentary canal was distinct and a streaming movement was observed from the anterior end to the anal end. Pigmentation was seen to extend from the cephalic to the caudal end. The anal aperture was well formed and distinct, closely placed with little inter orbital space. At 144 hrs the larvae measured an average length of 3.92 mm and showed deeper pigmentation dorsally and laterally. At 160 hrs the larvae measured 4.59 mm (Plate 3 E). The alimentary canal was vivid and clear, 90 % of the yolk reserve was consumed and vestiges of the pectoral fins appeared as finbuds. At 172 hrs the larvae were deeply pigmented. The body assumed a fish like shape with a well formed caudal fin and rudimentary pectoral fins (Plate 3 F). The larvae started to swim actively and were observed to feed voraciously. Details of larval development with the corre- sponding time scale is given in Table 2.

Discussion The diameter of the fertilised eggs of

M. cephalus as reported in earlier studies is seen to vary from 0.48 - 0.80 mm, in different environmental profile by Chaudhuri e t al. (1977), Ling (1970) and Kuo e t al. (1973). In the present study the spawned eggs ranged in diameter from 750-760 pm at a tem- perature of 26-28°C and salinity of 26%0. In a series of spawning experiments during 1988-'90 Tamaru et al. (1994); Kuo e t al. (1973) and Nash et al. (1974) observed that the diameter of spawned eggs ranged from 863.9 +. 20.7 pm - 938.6 * 32.1 pm in M. cephalus.

Embryonic development through the critical stages of cleavage, blastulation,

gastrulation and neurula stages with the corresponding time scale as ob- served in the present study was seen to be almost similar to that reported by Tamaru et al. (1993). The fertilised eggs have a diameter of 770-778 pm after 24 hrs of fertilization. The embryo showed deep pigmentation and twitching move- ments at regular intervals. At 28 hrs after fertilisation the embryonic heart beat could be observed. Kuo et aL. (1973) reported that in M. cephalus the heart began to beat 25.10 hrs after fertilisation.

Two environmental factors viz., tem- perature and salinity have profound influence on the development and hatch- ing of marine teleost eggs (Blaxter, 1998). In M. cephalus it has been dem- onstrated by Walsh et al. (1991) that salinity does not influence the time of hatching in this species. However, the time of hatching is inversely proportional to the incubation temperature. Hatching time has been observed to vary from 28- 54 hrs in different salinity-temperature combinations in studies made by Kuo et al. (19731, Liao (1975), Rajyalakshmi et al. (1991) and Krishnan et al. (1996). In the present study first hatching was observed at 30 hrs and hatcE-~g was completed at 32 hrs at a temperature of 26-28°C and salinity of 26 ppt. Tamaru et al. (1993) observed hatching at 28 hrs at a temperature of 26°C.

Eda et al. (1990) reported that the mean total length of the newly hatched larvae was 2.68 + 0.06 mm. In the present study the newly hatched larvae had a mean length of 2.29 mm. The mouth was observed to open on the second day and was functional on the third day post-hatch. Rajyalakshmi et al. (1991) reported that the larvae had developed mouth on the third day and was h c t i o n a l on the fourth day. Eda

Embryonic and larval development of Mugil cephalus 129

Plate 3. A. 72-hr old Iarve, B. 96-hr old larva, C. 108-hr old larva, D. 144-hr old larva, E. 160-hr old larva, F. 172-hr old larva. (Magnification: x 10).

e t al. (19901, Kuo et al. (19731, Nash e t al. (1974) and Liao et al. (1971) observed that the mouth of the mullet larvae open and yolk absorption is completed by the second and fourth day post-hatch respectively. Hence, as in the present study the most critical period in the rearing of M. cephalus larvae coincides with the opening of the mouth i.e. the secondfthird day post-hatch. Apparently active feeding on rotifers by the larvae begin before the completion of yolk sac absorption and it is vital and imperative that food organisms should be presented

to the mullet larvae by the second day, 36 hrs post-hatch. At 96 hrs the mouth opening measured 135 pm and has a direct bearing on the kind of rotifer fed to the larvae with reference to size. Brachionus plicatilis, the most com- monly used larval feed for M. cephalus may be of the L or S type and is observed to have a lorica length that varies from 110-340 pm. The small size of the larvae represents an even smaller mouth size at the time of first feeding. Fresh water fish larvae, in general not only hatch out at a larger size (ie. possess a largec

Mathew Abraham et al. 130

TABLE 2. Larual development of Mug11 cephalus

Time after Description Plate hatching

0 hr. Just hatched embryo, transparent well defined yolk 2 A sac, with a transparent fin fold encircling the body. Total average length 2.29 mm, head length 0.22 mm maximum width 0.81 mm.

24 hr. Average length 2.65 mm; dark prominent eye spot on 2 B the anterior part of head. There appears a break in the finfold ventrally with the anal aperture beginning to form.

72 hr. Well defined mouth opening and jaw movement 3 A observed. Larva has a long tapering tail and exhibits free movements.

96 hr. Larvae having an average length of 2.80 mm; mouth 3 B opening 135 pm, 60% of yolk utilised. Alimentary canal distinct.

108 hr. Larvae free swimming and have an average length of 3 C 2.84 mm. Eye balls large and distinct.

144 hr. Larvae measure an average length of 3.92 mm and 3 D show deep pigmentation.

160 hr. Rudimentary pectoral fins and well defined 3 E myomeres appear.

172 hr. Larvae deeply pigmented, body assumes a fish like 3 F shape with a well formed caudal fin and rudimentary pectoral fms visible.

mouth) but are morphologically more advanced than the marine teleosts a t first feeding. This allows for the exclu- sive use of artificial diets in their rearing. In contrast, marine fish larvae have to rely on live food organisms as their initial foodstuffs (Tamaru et al., 1993). This fact poses to be the most difficult and critical in sucessful larval rearing of mullet larvae. The present study, with special mention on its feeding on the embryonic and larval development of M. cephalus for the first time in India, will help refine the present level of knourledge on induced breeding and larval rearing.

Acknowledgment

The authors are grateful to Dr. K. Alagarswami former Director and to Dr. G. R. M. Rao, Director for their encouragement and help during the course of this study.

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