Indian Journal of Marine SciencesVol. 5, June 1976, pp. 113-116

Aquaculture of Green Mussel Mytilus viridis L. : Spawning,Fertilization & Larval Development

K. VIRABHADRA RAO, L. KRISHNA KUMARI & S. Z. QASIM

National Institute of Oceanography, Dona Paula 403004

Received 20 December 1975

Ripe mussels were induced to spawn under laboratory conditions by the application ofthermal, mechanical and chemical stimuli. Spontaneous spawning was observed in boththe sexes in summer months. The early cleavage of the fertilized eggs was traced and diffe­rent stages of the larvae up to the settling stage were successfully reared in the laboratory.The larval characters were described for the first time to enable the identification of planktonicmussel larvae from the natural environment. In M. viridis, the rate of larval development wasmuch faster than in the European species, M. edulis or in the Japanese species, M. crassitesta.The algal cultures of Tetraselmis gracilis and T. chui were used for feeding the developinglarvae.

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MYTILUS VIRIDIS is an important bivalvemollusc exploited for foed in India a:r:d inseveral other parts of the Indo-Pacific

region. Its rate of growth, breeding rericdicity,seasonal abundance of the larvae in the environ­ment and size composition of ce.mmercial catcheshave been describedl. The present paper is tr.efirst in the series on aquaculture and deals with n.espawning behaviour, early develofment, ctargesin the larval characters through various stages andthe culture techinques employed for rearirg n.elarvae in the laboratory up to the settling stage.The success achieved with this species appears tobe most promising for initiatiLg a large ~cale prcduc­tion of larvae and spats for culture operations.Moreover, this study gives the correct descriptionof all the larval stages of the species which is esstn­tial for the assessment of spatfall and populationdensity. Such an information is lacking in thelit erature.

Materials and MethodsMussels were obtained from the intertidal rocky

areas of Goa. They were placed in rectangula"rglass tanks containing sea water which was changedevery day. The mussels were fed on unialgal cul­tures of Tetraselmis gracilis, T. chui and Synecho­cystis sp. Batches of 3 to 4 mussels were takenat a time for inducing thEm to spawn. Thermal,mechanical and chemical stimuli were triEd withvarying success. In the spawned eggs, fertilizatienoccurred in finger bowls, when they were expo~edto milt obtained from the males. The fertilizedeggs and developing larvae were periodically remov­ed with the help of a pipette and examined u:r:der amicroscope. Each stage was figured under a cameralucida. For changing the water from the fingerbowls, the container was gently shaken in a cir­cular fashion and the larvae accumulating at thecentre were transferred with a pipette to a freshfinger bowl containing millipore-filtered sea water.

A few ml of Tetraselmis culture were added totte finger tGwls after the daily change of water.The remainirg water in the finger bowls, aftertLe removal of the larvae, was not rejectedtut col!eded in a larger jar to recover the remain­ing larvae. larvae frcm the larger jar were perio­dica]]y nfEcV(d ty filterirg the water througheoiting nylen (to [lm me,h width) and hpt in fresh,ea water with algal culture. Wten the larvaedevekped pi£mented eye srots, different ~ettlingffiaterials were placed in the finger bowls to inducettem to ,eitle.

SPauning - M. viridis was reported to breedalorg n_e Goa ceast a:most throughout the yearwith 2 peaks of spawning - one from Septemberto NOYEm1::erand, he other frem February to MarchI.Mussels with ripe gonads were stimulated to spawnby raising the temperature of sea water from 26·5­28°C to 32-35°C in the laboratory. Mussels keptin sea water with higher temperature spawnedwithin 30 min. The males spawned more readilythan the females. The eggs thus obtained werehealthy and their fertilizaticn was velY ~atisfaetolY.The males spawned very quickly when the isolatedgonads of females were kept in warm water in whichthe mus~els were alreaciy present. However, theadditie,n of spelms to the medium did not stimulatethe females to spawn except on a single occasion.

Tl-.e European mus~el M. edu/is was reportedto sr;awn ty peggirg it with a woeden wedge andthrety givirg it a mectanical stimulus or by prick­irg tte addutN II_mele with a needle in sea waterhaving a raised temperature of 19°C (ref. 2). Thismethcd on M. viridis was without much successalthol'gh a few eggs at times were set free. Partialsuccess was obtained by injecting 1% aqueoussolution of potassium chloride or liquor ammoniainto the mantle edge but such eggs were avoided farfurther sh:dy because of chemical contamir.ation.

Eggs ard Eperms obtained frcm spontaneousspawnings in the laboratory were found to be best

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INDIAN J. MAR. SCI., VOL. 5, JUNE 1976

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Fig. 1 - Egg showing the vitelline membrane and yolkgranules. Fig. 2 - Egg surrounded by sperms. Fig. 3­Extrusion of the first and second polar bodies. Fig. 4­First cleavaged egg showing the trefoil appearance. Fig. 5- Two celled stage showing difference in the size of cells.Fig. 6 - Formation of the second polar lobe. Fig. 7 - Secondcleavage and formation of 4 celled stage. Fig. 8 - Eightcelled stage. Fig. 9 - Blastula stage. Fig. 10 - Trochophorestage. Fig. 11 - Smallest straight-hinge stage with welldev6loped velum having the long cilia. Fig. 12 - Veliger

(24 hr old) showing the appearance of muscle strands.

of valves 58 [Lm. Slender velar retractors originat­ing from the hinge were clearly seen and theseextended up to the velum. Parts of the alimentarycanal were not clearly differentiated yet (Fig. 12).

At this stage, the larva has a flagellum apparentlyfor tactile functions (Figs 11 & 12).

After. 48 hr, the veliger grew to 88 [Lmin length,65 [Jomin valve height with the hinge remainingabout the same as in the earlier stage (64 [Lm).The anterior adductor muscle was well formed andthe alimentary canal showed an enlarged stomach,pale green digestive glands and a coiled intestine,ending into rectum. Particulate material compris­ing food ingested by the larva was seen movingwithin the stomach by the ciliary action of theepithelial lining (Fig. 13). From this stage onwards,the larvae were fed on the culture of Tetraselmisgracilis.

The 5 days oldlarva continued tobe in the straight­hinge stage, showing better developed structures:The valves increased to 120' [Lm in length and

suited fbr fertilization; and further developmentwent on satisfactorily till healthy larvae were hatch­ed. From January to April 1975, spawning occur­red a few days before or a few days after the fullmoon days. Males spawned in white streaks ofmilt which quickly dispersed in the surround­ing water making it somewhat cloudy. Femalesalso liberated the eggs in streams but these sankto the bottom of the finger bowls as a reddish patchclose to khe spawning mussel.

Segmentation of the fertilized eggs and formationof trochrJphore - The spawned eggs of M. viridiswere spherical (45 to 50 [Lm diam.) with reddishorange ~olouration filled with translucent yolkgranules and surrounded by a thin transparentvitelline membrane (Fig. 1). Fertilization readily

occurred] when they were surrounded by spermato­zoa (Fig 2), and within 30 min, the 1st and the 2ndpolar bodies were extruded (Fig. 3). The formationof the polar lobes is a characteristic feature of theeggs of iMytilus sp., Ilyanassa sp. and Dentalium

Sp.3,4. ~ M. viridis, the 1st pol~r lobe aPJ?earedalong wIth the 2nd polar body (FIg. 3). ThIS wasfollowed by the 1st cleavage which divided the egginto AB fnd CD cells. The round polar lobe remain­ed attac~ed to the CD cell giving the egg a trilobedor 'tref9il' appearance (Fig. 4). Finally the 1stpolar lODe was absorbed into the CD cell, making

this celli bigger than the AB cell (Fig. 5). Justbefore tHe 2nd cleavage, the 2nd polar lobe appear­ed (Fig. :6) and 4 cells A, B, C and D were formed{Fig. 7)'1 The 2nd polar lobe retracted into theD cell. When the 3rd cleavage which was spiral,took place, the 3 cells A, Band C divided equally,each re$lting into 2 small cells. However, Ddivided ]lnequally (Fig. 8). Subsequent cleavagesresulted in smaller cells spreading over the dividedlarger cell (Fig. 9), giving rise to the blastula stagewhich deiVelopedcilia and began to rotate. Gastru­lation Jook place by epiboly and the blastoporeappeared at the vegetative pole. Later on, it shiftedto the v¢ntral side where the stomodaeal pit deve­loped. Subsequently, the archenteron was formedand the embryo became elongated, broad at theapex and acquired a tuft of a few elongated cilia.n also Cleveloped a shell gland which began tosecrete the elements of a shell. Thus a fairly acti­vely mov~ng trochophore measuring 58 [Lmin lengthappeared: in about 6 hr after fertilization (Fig. 10).

Early land: late veliger stages - Rudiments of ashell seelt, in the trochophore got enlarged in 18 hrafter feIjtilization to form the 2 valves hingeddorsally., The hinge-line was straight and hence

designat~d as the 'straight-hinge stage'. The velumwas fair1~ well developed with the central regionhaving 2! enlarged cilia from the apical tuft of thetrochophore. The archenteron was present as an

irregular Isac· with no mouth, intestine, rectum oradductor muscle. The smallest of· the straight­hinge veligers was 73 f~min length, with a hinge62 pomlong and 50,9 [Lmhigh valve (Fig. 11). Thevehger m,ade active movements but no feeding hadcommenaed.

At the ~nd of 24 hr the veliger measured as follows:length of valves 76 p.m, hinge 63 fJ.mand height

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RAO et at.: GREEN MUSSEL: EARLY DEVELOPMENT

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fibres, ground-glass and plain glass bits and pebblesof stones were suspended or laid at the bottom ofthe finger bowls to persuade the larvae to settle with­out success. While larval rearing was a successfulevent in these initial experiments, no settlementoccurred. However, on one occasion, a spat wasfound attached to the wall of a jar from which thewater was drained off periodically into finger bowls.The spat was alive but an attempt to remove itfrom its place of attachment damaged the specimen.This broken piece showed well-fonned gill filamentsand labial palps; the broken part of the shell wasyellowish in colour.

Another spat collected on the 19th day was inan inactive state and measured 388 [J<min length(Fig. 18). It had the appearance of seed musselsseen in the natural environment. Its umbo wasfairly prominent and the shell had close-set concen­tric lines of grovrth. The internal structures couldnot be studied as it died with the valves tightlyclosed. A few pediveligers were isolated and reared

Fig. 13 - Veliger (48 hr old) with well formed anterioradductor muscle, intestine. digestive gland. etc. Fig. 14­Nine days old veliger with stomach, statocyst, foot, andlabial palps. FigS. 15 and 16- Fourteen days old larvae.Fig. 17 - Sixteen days old larvae with well formed eyespot.Fig. 18 - Spat on 19th day [Abbreviations: ada, anterioradductor muscle; adp, posterior adductor muscle; dg, diges­tive gland; ey, eye; ft, foot; gl, gill; int, intestine; lp, labialpalp; st, stomach; stc, statocyst; ve, velum; and vr, velar

retractors]

-81 (J.m in height. By the 7th day, the larvae were132 fLmin length and 106 fLll1in height.

On the 9th day, the largest larva measured 151[Lmin length and 126 [Lmin height of the shell valve(Fig. 14). The umbo was not yet formed but therewas further development of soft parts. Theposterior adductor muscle and the statocyst weredearly seen. The labial palps, foot and the pri­mordium of the gill had developed. The larvaeas before were swimming in water at all levels frombottom to the surface of the finger bowls.

Fourteen days after the ftortili~ation, a wel1­defined umbonal stage veliger was noticed for the1st time, measuring 225 fLmin length and 226 fLmin height and 149 fLmin thickness (Figs. 15 and 16).In addition to the structures already described inthe earlier stage, it also possessed a dark pigmentedeye spot (hence known as the eyed larva). Thewell-developed velum was still present, but themovements of the larvae were largely confined tothe bottom.

It is interesting to note that the larval develop­ment was not uniform even in the same ba'.ch ofeggs. When the eyed larvae appeared in the culturebowls after 14 days, some still remained small andstunted, measuring no larger than the 3 day oldlarvae. Retardation in growth in these was notdue to the lack of food, for most of the larvae surviv­ed very well on Tetraselmis gracilis which was sup­plied in sufficient quantities every day.

The 16 days old larvae measured 278 fLll1in lengthand 260 fLmin height (Fig. 17). These had all thestructures noted earlier. The posterior adductormuscle (adp) had grown much larger than theanterior adductor muscle (ada). The velum (ve)was still the only locomotor organ possessing ciliaand it showed no sign of reduci.ion yet. Thepigmented eye spot became conspicuous (ey). Thefoot (ft) was a little larger than the earlier stagebut was not used for creeping. The labial palps{lp) were large and the gill on each side had a 3­lobed appearance. The general colouration wasyellow, the umbonal region became darker in appear­ance and the digestive gland was greenish brown.Some of the veligers showed a pale pinkish coloura­tion near the umbo both at this and in the previousstages. The shell showed some concentric striationsindicating the growth lines.

Pediveliger and spat -The period following 16thday was marked by almost no further change.A few larvae, however, grew to about 300 fLminlength and developed a slightly oblique shell anda finger-like foot which often protruded and retract­ed between the partially gaping valves. The larvaeattempted to secure attachment after slow creep­ing movement on the substratum. However, thisstage may not be regarded as the pediveliger stageseen in most bivalves. The larvae had a slightlyreduced velum and their locomotion was partlyby swimming with the help of the velum and partlyby creeping at the bottom of the finger bowls by theslender foot. Occasionally, a weak byssal attach­ment was noticed, but the larvae were not firmlyTIxed. Several types of culch materials, namelyshell pieces of oysters and mussels, slender rope

INDIAN J. MAR. seI., VOL. 5, JUNE 1976

separately in finger bowls, changirg the water dailyand supplying them with Tetraselmzs culture. Theycrept actively at the botte-m using their foot andoccasionplly swam with their veh:m which wasstill preiSent. They did net undergo any furtherchange. The last of the pediveligers died on the56th da)!' after the fertilization of eggs.Discussion

Sea m~ssels aJl over the world fmm one of theimportant food resources. In Irdia, the need forculturing them to augment r.atural prcductionhas been emphasized by Qasim and AC'tari5 andRao et aU. Attempts to culture them by coEedingthe seed~ from the natural envirorment a:r:drearingthem on (floatirg rafts or on stakes are alrEady under­way. These methods give exceedirgly high yieldsin tropical water~6. Thus the availability of largequantiti$ of seeds is an essential prerequisite forculturing] them on a commercial scale. Seed pro­duction lunder controned conditic-rs by irducingthe adults to spawn and then rearirg the larvaefrom fertilized eggs to the settling stage, form animportant aspect of large-scale cuaure operatic-noThe stimuli that induce spawning, H.e algal culturessuitable lor larval rearing, tolerance ar.d optimalconditions of the medit:m for the survival of larvaeand the special c1:aemotactic requirements whichpromote settlfment of the larvae are brcadly k:r:c,','i!lfrom thel earlier works2,7-9 .• Yet, the requirementsof the mussel seem to be so varied frcm region toregion that they have to be determined by furtherexperime,Vtation with each species in different geo­graphical, areas.

In any cultivable species, stimulaticn for spawningis an important factor to obtain eggs and spermsin a healthy condition. When the present investiga­tion was started, all that was k:r:own of inducedspawning in M. viridis, was the laboratory trialscarried out in Singapore, which had met with limitedsuccess10, but the exact nature of stimulation usedwas not known. In this communication onlythe preliIl}inary findings have been reported. Thereis much scope for detailed observatic-:r:son a varietyof chemicals to be used for ind1.:Ced~pawning inbivalves and also of electrical stimulation whichhad not been attempted so far.

In feeding the mussel larvae, Chlorella, Tetraselmisand Synechocystis were tried. Chlorella was usedwhen it was grcwing very slc.wly in wCcultures.However, Loosanoff and Davis2 have successfullygrown th~ larvae of M. ed~tlt's on an algal mixtureof Chlorella and other green forms. Tetraselmisgracilis and T. chui grtw fast in au subcultures,and hence these were effectively used. Synecho­cystis alsol grEW fast in sue cultures but the larvae

remained stunted when they were fed on this algaalone. Growth of the larvae was fairly good whenthey were fed on a mixture of Synechocystis andTetraselmis gracilis. This mixture was found suit­able for feeding the adult mussels kept in thelaboratory.

A large variation in the growth rate occurredamong the larvae when the eggs of one female werefertilized by the sperms of different males. This.agrees well with the findings of Loosanoff and Davis2in M. edulis. The larval development in M. viridiswas faster than either in M. edulis or in the Japanesemussel, M. crassitesta. In M. viridis the straight­hinge stage was noticed within 16 hr after the ferti­lization aLd the umbo stage within 14 days in thelaboratory with salinities rangirg frem. 34 to 35·6%0­and temperature from 25° to 27·4°C. Probably afaster grcwth occurs in the natural environment.In M. edulis, the smallest straight-hinge stage isreported to reach within 48 hr3 ard in M. crassitestathe straight-hinge stage in 3 cays and the umbostage in g6 cays4. A faster rate of grcwth in larvaldevelorment is conducive to tatc:hery operations.

It is kLcwn that ciliates, eactelia aLd fungi takea heavy toll of the larvae rarticularly after thefirst fE.w days of rearirg as observed earlier10,1I.In the firger hwls, t:he la1val mortality was lew butin the large (4 1) jars, in which the water was renew­ed less frequOllly, t:tH~ciliates multiplied fast, result­ing in greater mortality of the larvae. Preventionof the growth of ciliates usirg a dilute solution offungicides can dtfinitely lead to a greater survivalof the larvae in the laboratory.

References1. RAO. K. VIRABHADRA,KUMAR!,L. K. & DWIVEDI, S. N.,

Indian J. mar. Sci .• 4 (1975) 189.2. LOOSANOFF,V. L. & DAVIS. H. c., Advances in marine

biology. Vol. I (Academic Press, London and NewYork), 1963. 1.

3. REVERBERI,G., Experimental em/;ryology of marine amIfreshu:ater tnvertebrates (North Holland Publishing Co.,Amsterdam, London), 1971, 182.

4. CAHN,A. R., Clam culture in Japan (G. Hd. Qrs, SupremeCommander, Allied Powers: Natural Resources, Report146), 1951, 70.

5. QAsnir, S. Z. & ACHARI,G. P. K.. Seminar on mariculturlJ'and mechanized fishing, Dept of Fisheries, Govt. of TamilNadu, 1972. AbstrClcts. 13.

6. QASIM, S. Z., Prec. Indian natn. Sci. Acad .• 41H­(1975), 397.

7. GALTSOFF,P. S., Biol. Bull., 78 (1940), 117.8. WALNE, P. R.. Physiology of mollusca. Vol. 1. edited by

K. M. Wilbur & C. M. Young (Academic Press. NewYork and London), 1968, 197.

9. hw, T.. in Coastal aquaculture in the Indo-Pacific region.edited by T. V. R. Pillai (FAO, IPFC). 1973,260.

10. Kow, T. A., LING, Y. S. & HIN, Y. T. W .• in Coastalaquaculture in the Indo-Pacific region, edited by T. V. R.Pillai (FAO, IPFC), 19i3. 374.

11. CULLINEY, J. L., Bioi. Bull., 147 (1974), 321.

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