Embryonic and larval development of the striped mullet Mugil ...

Embryonic and larval development of the stripedmullet Mugil eephalus CL)MATHEW ABRAHAM, P. SHIRANEE, P. KISHORE CHANDRA, M. KAILASAMAND V.K. CHARLESCentral Institute of Brackishwater Aquaculture, Madras-600 008, IndiaABSTRACTMugil cephalus in ripe condition collected from inshore catches at Muttilkadunear Madras were induced to breed using HCG and ovaprim to study theembryonic and larval development. Samples of unfertilised eggs and variousdevelopmental stages from fertilization through embryonic and larval stageswere preserved for further micrographic studies. Rate of fertilization wasdetermined at the time of blastodisc formation and was 90 %. Of the totalnumber of 1.46 million eggs, 1.26 million were fertilised. The pre-hatchedembryo was deeply pigmented, exhibited twitching movements and lay curvedover the yolk mass. After an incubation period of 30-32 hrs, the embryo hatchedout. The newly hatched out larvae measuring 2.29 mm, were transparent witha large oval head, a well defined yolk sac and short tail encircled by acontinuous finfold. hlouth formation was complete and the larvae startedfeeding on 3rd day of hatching. The paper presents the results of detailedstudies of embryonic and larva1 development and the time scale establishedfor critical stages of development.IntroductionEmbryonic and larval developmentstudies besides providing interestinginformation in itself, are imperative andconsequential to the successful rearingof larvae for seed production. Despitethe successes in artificial propagation ofthe mullet, by induced spawning, thereis still a need to refine further thetechniques of larval rearing particularlyfor practical and commercial applications(Liao, 1993). It is an undisputedfact that larval rearing remains themost critical and crucial phase inbrackishwater fish seed production.Development of suitable protocols forthe mass rearing of larval fish repre-sents one of the last bamers for thesuccessful propagation of a variety ofmarine species (Tamaru et al., 1993).Most problems arise from relativelysmaller size of the mouth and limitedyolk reserves (Shirota, 1970) of thelarvae.The present study elaborates withphotographic evidence the embryonicand larval structure and developmentwith the corresponding time scale of M.cephalus. A photographic presentationof the embryonic development of M.cephalus with time scale has beenelaborated by Tamaru et al. (1993).Vivid photographs, however, with notextual reference have been made by

Mathew Abraham et al.Liao (1993). Earlier studies on thedevelopment of the mullets, conductedon various geographic and environrnen-/tal profiles include that of Nair (1957),:Anderson (1958), Kuo et al. (1973), Ling;(1970) and Chaudhuri et al. (1977).iMaterials and methodsiMugil cephalus breeders were ob- \ The total number of eggs was estimated-&-?&1.4 m and the rate of fertiliz 'ontained from commercial sea catches at 90 %. Incubation period spann / over aMuttukadu, near Madras during Febru- period of 30-32 hrs and the hatchingary 1997. Females with an average rate was 41.6 %. Immediately aReroocyte diameter of >525pm and milting fertilization the germplasm migrated tomales were selected. The selected fish the animal pole to form a dense cap likewere given a dip treatment in 1 ppm structure called the germinal disc (Plateacriflavin and 1 hr bath in 10 ppm 1B). Cleavage (Plate 1 C,D,E,F) wasoxytetracyclin. Induced breeding was restricted to the germinal disc and thedone following the two injection protocol resulting blastoderm assumed a discwith a 24 hr interval. HCG was used as like multi-cellular structure called thethe priming dose @ 6,000-10,000 I.U./kg blastodisc, which was more or lessbody weight and ovaprim was used as convex in shape and enclosed betweenthe resolving dose @ 3-5mIJkg body itself and the uncleaved residue of theweight. The males were not given any egg - a cavity representing the blastohormonetreatment. Dry stripping was coele (Plate 1G) cleacage was followedresorted to and fertilization was effected by gastrulation which converted thein-vitro. Incubation was done @ 140 embryo into a two layered structureeggsA. Water temperature ranged from (Plate I H). The outer germ layer, the26-28°C and salinity was 26 ppt. Sam- epiblast, gave rise to the ectoderm whileples of eggs before fertilization, imme- the involuted cell mass, the hypoblast,diately after fertilization and there- the endodermal and mesodermal comafterat 30 minute intervals until hatch- ponents.ing and including the larval samples at30 minute intervals were also preservedin a medium made of 2 % formalin, 4 %glycerol and 94 % water for furtherstudies. The larvae were reared in FRPtanks provided with flow through runningwater system at a density of 45-50nosll. The larvae were fed with therotifer Brachionus plicatilis from thirdday onwards.ResultsThe ovulation was complete 22 hrsafter the resolving dose. The strippedeggs were translucent and non adhesivewith a deep yellow colour. The diameterof the spawned eggs ranged from 750-760 pm. The yolk was characterised bya single yolk globule and the eggs weretelolecithal in nature (Plate 1 A). Fertilizationwas effected by stripping miltfrom males and mixing it with the eggs.Subsequent to gastrulation, developmentcontinued until a primitive vertebratebody was formed and the neuralplate established. The embryo was thenmore or less cylindrical and bilaterallysymmetrical and was referred to asneurula (Plate 1. I). The head andpharyngeal region projected from theyolk mass anteriorly and the trunkcurved over the yolk and the tailprojected posteriorly (Plate 1. J). Themesoderm on both sides of the notochordorganised into somites andmelanophores began to appear (Plate 1K). The pre-hatched embryo showed

Embryonzc and larval development of hlugil cephalus 125Platc: 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 127Plate 2. A. Newly hatched larva, B. 24-hr old larva. (Magnification: x 100).

Mathew Abraham et al.length of 2.80 mm and 60 % of the yolkwas utilised. The mouth opening measured135 pm in the 96 hrs old larvae(Plate 3 B). The alimentary canal wasdistinct and a streaming movement wasobserved from the anterior end to theanal end. Pigmentation was seen toextend from the cephalic to the caudalend. The anal aperture was well formedand distinct, closely placed with littleinter orbital space. At 144 hrs the larvaemeasured an average length of 3.92 mmand showed deeper pigmentationdorsally and laterally. At 160 hrs thelarvae measured 4.59 mm (Plate 3 E).The alimentary canal was vivid andclear, 90 % of the yolk reserve wasconsumed and vestiges of the pectoralfins appeared as finbuds. At 172 hrs thelarvae were deeply pigmented. The bodyassumed a fish like shape with a wellformed caudal fin and rudimentarypectoral fins (Plate 3 F). The larvaestarted to swim actively and wereobserved to feed voraciously. Details oflarval development with the correspondingtime scale is given in Table 2.DiscussionThe diameter of the fertilised eggs ofM. cephalus as reported in earlierstudies is seen to vary from 0.48 - 0.80mm, in different environmental profileby Chaudhuri et al. (1977), Ling (1970)and Kuo et al. (1973). In the presentstudy the spawned eggs ranged indiameter from 750-760 pm at a temperatureof 26-28°C and salinity of 26%0.In a series of spawning experimentsduring 1988-'90 Tamaru et al. (1994);Kuo et al. (1973) and Nash et al. (1974)observed that the diameter of spawnedeggs ranged from 863.9 +. 20.7 pm -938.6 * 32.1 pm in M. cephalus.Embryonic development through thecritical stages of cleavage, blastulation,gastrulation and neurula stages withthe corresponding time scale as observedin the present study was seen tobe almost similar to that reported byTamaru et al. (1993). The fertilised eggshave a diameter of 770-778 pm after 24hrs of fertilization. The embryo showeddeep pigmentation and twitching movementsat regular intervals. At 28 hrsafter fertilisation the embryonic heartbeat could be observed. Kuo et aL. (1973)reported that in M. cephalus the heartbegan to beat 25.10 hrs after fertilisation.Two environmental factors viz., temperatureand salinity have profoundinfluence on the development and hatchingof marine teleost eggs (Blaxter,1998). In M. cephalus it has been demonstratedby Walsh et al. (1991) thatsalinity does not influence the time ofhatching in this species. However, thetime of hatching is inversely proportionalto the incubation temperature. Hatchingtime has been observed to vary from 28-54 hrs in different salinity-temperaturecombinations in studies made by Kuo etal. (19731, Liao (1975), Rajyalakshmi etal. (1991) and Krishnan et al. (1996). Inthe present study first hatching wasobserved at 30 hrs and hatcE-~g wascompleted at 32 hrs at a temperature of26-28°C and salinity of 26 ppt. Tamaruet al. (1993) observed hatching at 28 hrsat a temperature of 26°C.Eda et al. (1990) reported that themean total length of the newly hatchedlarvae was 2.68 + 0.06 mm. In thepresent study the newly hatched larvaehad a mean length of 2.29 mm. Themouth was observed to open on thesecond day and was functional on thethird day post-hatch. Rajyalakshmi etal. (1991) reported that the larvae haddeveloped mouth on the third day andwas hctional on the fourth day. Eda

Embryonic and larval development of Mugil cephalus 129Plate 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).et al. (19901, Kuo et al. (19731, Nash etal. (1974) and Liao et al. (1971) observedthat the mouth of the mullet larvaeopen and yolk absorption is completedby the second and fourth day post-hatchrespectively. Hence, as in the presentstudy the most critical period in therearing of M. cephalus larvae coincideswith the opening of the mouth i.e. thesecondfthird day post-hatch. Apparentlyactive feeding on rotifers by the larvaebegin before the completion of yolk sacabsorption and it is vital and imperativethat food organisms should be presentedto the mullet larvae by the second day,36 hrs post-hatch. At 96 hrs the mouthopening measured 135 pm and has adirect bearing on the kind of rotifer fedto the larvae with reference to size.Brachionus plicatilis, the most commonlyused larval feed for M. cephalusmay be of the L or S type and is observedto have a lorica length that varies from110-340 pm. The small size of the larvaerepresents an even smaller mouth sizeat the time of first feeding. Fresh waterfish larvae, in general not only hatch outat a larger size (ie. possess a largec

Mathew Abraham et al. 130TABLE 2. Larual development of Mug11 cephalusTime after Description Platehatching0 hr. Just hatched embryo, transparent well defined yolk 2 Asac, with a transparent fin fold encircling the body.Total average length 2.29 mm, head length 0.22 mmmaximum width 0.81 mm.24 hr. Average length 2.65 mm; dark prominent eye spot on 2 Bthe anterior part of head. There appears a breakin the finfold ventrally with the anal aperturebeginning to form.72 hr. Well defined mouth opening and jaw movement 3 Aobserved. Larva has a long tapering tail andexhibits free movements.96 hr. Larvae having an average length of 2.80 mm; mouth 3 Bopening 135 pm, 60% of yolk utilised. Alimentarycanal distinct.108 hr. Larvae free swimming and have an average length of 3 C2.84 mm. Eye balls large and distinct.144 hr. Larvae measure an average length of 3.92 mm and 3 Dshow deep pigmentation.160 hr. Rudimentary pectoral fins and well defined 3 Emyomeres appear.172 hr. Larvae deeply pigmented, body assumes a fish like 3 Fshape with a well formed caudal fin andrudimentary pectoral fms visible.mouth) but are morphologically moreadvanced than the marine teleosts atfirst feeding. This allows for the exclusiveuse of artificial diets in theirrearing. In contrast, marine fish larvaehave to rely on live food organisms astheir initial foodstuffs (Tamaru et al.,1993). This fact poses to be the mostdifficult and critical in sucessful larvalrearing of mullet larvae. The presentstudy, with special mention on itsfeeding on the embryonic and larvaldevelopment of M. cephalus for the firsttime in India, will help refine thepresent level of knourledge on inducedbreeding and larval rearing.AcknowledgmentThe authors are grateful to Dr. K.Alagarswami former Director and toDr. G. R. M. Rao, Director for theirencouragement and help during thecourse of this study.ReferencesAnderson, W. W. 1958. Larval development,growth and spawning of striped mullet(Mugil cephalus) along the SouthAtlantic coast of the United States.Fish. Bull. US. Fish. Wild. Serv., 58(144) : 510-519.Blaxter, J.H.S. 1988. Pattern and variety indevelopment. In : W.S. Hoar and D.J.Randall (Eds.), Fish Physiology, Vol. 11,Part A. Academic Press. San Diego,p. 1-58.Chaudhuri, H., R.M. Bhowmic, G.V. Kowtal.M.M. Bagchi, R.K. Jana and S.D.Guptha 1977. Experiments in artificialpropagation and larval development ofM. cephalus (Linnaeus) in India. J.Inland Fish. Sac. India., 9 : 30-41.Eda, H., R. RIurashige, Y. Oozeki, A.Hagiwara, B. Eastham, P. Bass, C.S.Tamaru and C.S. Lee 1990. Factorsaffecting intensive larval rearing instriped mullet, Mugil cephalus.Aquaculture, 91 : 281-294.

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