Inducement of polyploidy in the Indian pearl oyster, Pinctada fucata (Gould).

/ ? r r E ’’ r ‘ ' c r y

INDUCEMENT OF POLYPLOIDY IN THE INDIAN

PEARL OYSTER, Pinctoda fucota (GOULD)

D I S S E R T A T I O N S V B M I T T L I ) B Y

M. SAMAYA KANNAN

I N P A R r i A L F U L F I L M E 1 \ T 0 F T H E R E Q U I R E M E N T S F O R T I I E D E G R F F O F

MASTER OF FISHERIES SCIENCE (MARICULTURE)

o r THE

CENTRAL INSTITUTE OF FISHERIES EDUCATION

(D!';i:m i;i) uNivr.RsiiY) VLUSOVA, MUMIJAl -400 061.

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INDIAN COUNCIL O F AGRICULTURAL RESEARCH

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N O V U M I3 L K , 1998.

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DECLARATION

I h e re b y declare that this thesis entitled “Iiuluceinent o f p o ly p lo id y in the Indian pear l oyster, PincUula f u c a t a (Gould) ” is based on m y o w n research a n d has not p re v io u sly fo rm ed the basis fu r the a w a rd o f any degree, diploma, assoclateship, fe llo w sh ip or other sim ila r titles or recognition.

Kochi, ( M . S A M A Y A K A N N A N )

November, 1998.

CERTIFICATE

C ertified that the d issertatio n entitled “liidiiccmciit o f p oly p lo id y in the Indian p c n i i oyster V inciada f u c a i a (Gould)” is a bonafide r e c o r d o f work done by Mr. M . S A M A Y A K A N N A N , under our g u id a n ce at the C e n tra l Marine Fisheries R esea rc h Institute d u rin g the tenure o f h is M.F.Sc. (M ariculture) programme o f 199 7 - 1999 and th a t it has not p re v io u s ly fo rm ed the b a sis fo r the award o f any o th e r degree, diplom a o r other sim ilar titles or fo r any publication.

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D r. D. B. JAMES, S e n i o r Scientist,

T R C o f C . M . F . R . I . , T u t i c o r i n ( M e m b e r , A d v i s o r y C o m m i t t e e )

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PREFACE

B ivalve molluscs su ch as edible o yste rs, pearl oysters, mussels, scallops, cla m s and cockles a re w id e ly distributed both in the tro p ic a l and tem perate w aters. Most bivalves a re exploited for th e ir delicious m e a t and as a cheap source o f protein. In re ce n t years, the d e p le tio n of wild s to c k s due to intensive e xp lo ita tio n and also p o o r productivity from the w ild due to environm ental pollution and d a m a g e , increased th e demand for fa rm grown products. U n lik e other bivalves, th e pearl o y s te rs are cu ltu red fo r their precious pearls and shells.

The w o rld aquaculture production o f p e a rl oysters h a v e been gradually d e cre a sin g for the p a s t fe w years to ta llin g 16,956 t in 1995 contributing n e a rly 0.33% of the to ta l marine m o llu s c production o f 5,093,747t (FAO,1997).

W orld production o f pearls is e stim a te d at betw een 80-100 tonnes/year. Ja p a n is the largest produ cer of p e a rls and the la rg e st importer o f raw pearls too. Eventhough Ind ia had su cce ssfu lly produced cultured pearls in 1973 itself, it could not p ro d u ce cultured p e a rls for world tra d e .

India supplied about 3 2 kg of marine p e a rls in 1989 fo rm in g only 0.49% of the to ta l pearl im ports o f Japan estim a te d at 6,512 kg (Seam us McElroy, 1990).

T h e pearl quality m a y be improved by th e application o f modern tools such as genetic im provem ent and tissue cultu re (A lagarsw am l, 1987;

V ictor and V elayu lhan , 199^). T h e application o f genetic m a nipu la tions like polyploidy, gyn oge nesis and androgenesis in aquaculture h a s received considerable a tte n tio n in recent years.

T rip lo id s and tetraploids can be produced by application o f thermal shock, p ressure shock or che m ical shock at just b e fo re meiosis a n d mitosis

o f fertilized e g gs respectively. A d u lt (riploids a re expected to b e sterile since they can no t synapse hom ologous chrom osom e d u rin g gam etogenesis.

T h e advantages of in d u c e d polyploidy a re ;

I. Energy utilized for g a m e te production is diverted to s o m a tic growth.

Therefore, adult triploid co u ld g ro w faster.

if. R educed gam etogenesis increases the d ry m e a t weight a n d prevents su m m e r m ortality which re su lts In fetching g o o d m arketability throughout the year.

ili. T riploidy enhance the p h ysiolog ica l activity lik e disease re sista n ce etc.

iv. T etraploid animals are u se d to produce c e n t percent via b le triplolds by crossing w ith normal diploids.

The induced triploid p e a rl oysters w ill b e a useful tool fo r shortening the period o f peari culture, im p ro vin g the yield a n d quality o f p e a rl (Lin and Jiang, 1993).

Induce m en t of p o lyp lo id y In pearl o yste rs is not at p re s e n t undertaken in India. A s the species o f p e a rl oysters a re econom ically im portant in terms o f pro d u ctio n of n a tu ra l and cultured pearls, the d e ve lo p m e n t of sterile oyter through p o lyp lo id y induction Is o f param ount im portance in peari p rodu ction. During cu ltu re d pearl production, the oysters w ith matured gonad m a y ham per the p ro c e s s of nucleus implantation a n d lead to

m alform ation of pearls.

Inducem ent of p o ly p lo id y may preven t gam etogenesis w hich pave the w a y fo r large-scale production o f high qu ality cultured pearls.

ACKNOWLEDGEMENTS

1 am ever grateful to S hri.S .D harm araj, Senior Scientist, TRC of C.M.F.R.I., Tuticorin, for his constant encouragem ent, g u id a n c e and supervision o f the present work. \ w ish to e x p re s s my sincere thanks to Shri.D .C .V.Easterson, Senior S cientist and O fficer-in-charge, T R C of C.IVl.F.R.I., Dr.D.B.Jam es, S e n io r Scientist and I a ls o extend my g ra te fu l thanks to Shri.A.C hellam , Dr.P.Muthiah and Shri. K .R am adoss, Senior S cie n tists for their valuable a d v ic e and for p ro vid in g lab fa c ilitie s for my w o rk. M y sincere thanks are a ls o d u e to D r.(M rs.)S h oji Joseph fo r h e r valuable suggestions and encouragem ent.

I w o u ld like to e x p re ss my d e e p sense o f g ra titu d e to Dr.C.Suseelan, Senior Scientist and O fficer-in-charge, PGPM, C .M .F .R .I., for the patience and tolerance s h o w n by him in understanding a n d helping to overcome th e problem s faced d u rin g the period o f the study. I exp re ss my deep gratitude to Dr.M .Devaraj, Director, C .M .F .R .I., for p ro vid in g all necessary fa c ilitie s at the In stitute, during the c o u rs e of this study.

I am thankful to D r.K.R engarajan, S e n io r Scientist, C.M.F.R.I., Kochi, and Dr. V . Ramadhas, Professor, F ish e rie s College a n d Research Institute, T u tico rin for their h e lp in sparing th e ir valuable time fo r correcting my dissertation. M y heartfelt th a n k s are also due to S hri.P.Jaw ahar, A sst. Prof., Fisheries C o lle g e and Research Institute, T u ticorin fo r his critical re a d in g of this manuscript. I convey my th a n k s to Miss. R o s a lie Shaeffer, Technical Information Specialist, U.S.A. a n d Dr.Lin Y ueguang, South China S ea Institute of O ceanology, People's R e p u b lic China, for th e ir immediate response in sending re fe re n ce s, reprints and com puter d iske tte s related to th is present work.

I am deeply indebted to Dr.N .Sukum aran, Prof. and Head, Shri Param akalyani Centre for Environm ental S ciences (S P C E S ), M.S.U., Afwarkurichi., and my h e a rtfe lt thanks are d u e to M r,J.G odw in, SRF, SPCES, M .S.U., for their h e lp rendered in taking photo gra phs of chromosomes.

I ta ke this opportunity to thank all the s ta ffs of PGPM, C.M.F.R.I., and the s ta ffs o f TRO of C .M .F .R .I, Kochi e sp e cia lly Mr.Shanm ugasundaram , Mrs.Su/a a n d Mr.Rocfrigo, the s k in divers M r.S ekar, M r.P.M uthukrishnan and Mr.Jesuraj fo r th e ir timely help and co-operation. I am a lso thankful to Mr.Joe and Mr.Muniasamy, M /s. Raja A gen cies, Tuticorin, fo r providing matured o yste rs during the re search.

M y special thanks a re d u e to my friend M r.A. Anand, S R F , C.I.F.E., for his valu able comments o n th e preparation o f chrom osom e spreads. My heartfelt thanks are due to M r. Ju liu s Edward, M r.T.A nand and M r.V .N .B iju and Mr.Jude, F.C. & R.I., Tuticorin, fo r th e ir generous help in this stud y. I owe a lot to m y friends, well-wishers, s e n io rs , classm ates and juniors fo r th e ir moral support and encouragement.

1 appreciate the n e a t and timely e x e c u tio n of this disse rta tio n by Mr. M uniasa m y and Mr. Selvan, M/s. Ves C o m p u te r Data P roce ssing Centre, Tuticorin, a n d rem ain thankful to them .

I am greatly thankful to m y brother M r.M .Sathiyavan, w h o shared most o f m y w o rk gleefully. M ere w o rd s would b e little for the in s p ira tio n support

and encourage m en t of my pa re n ts, sisters M uthu, V asuky and S a ra th a .

I acknowledge the In d ia n Council o f Agricultural R ese arch , New Delhi fo r a w a rd in g the Junior R esearch Fellow ship throughout th e period of my M.F.Sc. co u rse .

CONTENTS

C H A P T E R TITLE Page

1. IN TR O D U C TIO N 1

2. REVIEW O F LITERATURE 4

3. M ATER IALS A N D M ETHO DS 18

A. RESULTS 24

5. DISCUSSION 36

6. SUM M ARY 44

7. R EFER EN C ES 46

ABSTRACT

ABSTRACT

Sam ayakannan, M.,1998. In d u ce m e n t of po lyploidy in the Indian p e a rl oyster, P in c ta d a fucata (G ould). M.F.Sc. dissertation, C e n tra l Marine F ish eries Research In stitu te . C.I.F.E. (D eem ed U niversity), Kochi.

(S.Dharm araj).

In In d ia n pearl oyster in d ustry, triploid s te rile pearl oyster w o u ld play a vital role as prolonged m a tura tion is one o f th e problems fo r nucleus implantation. In this present investigation, an e ffe ctive method o f inducing polyploidy in th e Indian pearl o yste r, Pinctada fu c a ta (G.) developed b y using thermal shock. T h e fertilized e m b ry o s were exposed to thermal s h o c k a t 30. 32, 35, 38 and 6 °C at 10, 20 and 5 0 m inutes after fertiliza tion , for a d u ra tio n of 10 and 15 m inutes fo r heal shock and cold shock respectively. Ploidy le ve ls were determined b y chrom osom e co u n ts from trochop ho re lan/ae. T rip lo id embryos were encoun tered in all treated group s. But no trip lo id or tetrap lo id embryos were recorded in th e control groups.

It b e c a m e explicit fro m the present s tu d y that the m o s t effective procedure o f in d u cin g triploidy w a s exposing the fe rtiliz e d eggs at Z 6 °C a t 10 min for the first p o la r body retention and at 20 m in fo r the second p o la r body retention, w h ic h resulted in the fo rm a tio n of 64.3 ± 2 ,6 5 and 74.3 ± 2 .3 3 % triploid embryos respective ly. When th e fertilized eggs w e re treated at 3 5 °C a t the end of 50 min p o s t fertilization, the h ig h e s t percentage (46.6 ±. 1.66%) o f tetraploid embryos c o u ld be recorded. O bvio u sly, thermal s h o c k appeared a s th e best low cost te ch n o lo g y fo r the com m ercial production o f triploid and te tra p lo id oyster larvae m e a n t fo r pearl culture.

IISITRODLCTION

1. INTRODUCTION

P e a rl is one o f the n in e gem s and the o n ly gem o b ta in e d from the

living organ ism . Since the daw n o f hum an civilization , pearl has b e e n an object o fa d o ra tio n .lt has been tre a su re d since long back.

F ish in g for pearls h a s been practised fo r several cen turie s in many parts o f the w o rld including In d ia . Occurrence o f a natural s p h e ric a l quality pearl from a w ild oyster is a m e re chance a n d it is not cost effective. The uncontrolled overfishing o f th e s e oysters led to th e destruction o f m o s t of the natural pearl oyster beds kn o w n in Tamil as "P a a rs " o f India. M oreover, the natural pearl oyster beds a re often destroyed by the increasing num ber of bottom traw lers. Finally, the p e a rl fishery c a m e to an end in Ind ia by 1961.

Since (hen, th e availability o f n a tu ra l pearl w as fo u n d scarce and a need came to develop an alternative te ch n o lo g y for the production of c u ltu re d pearls.

Hence the C en tral Marine F is h e rie s Research Institute (CM FRI) had started pearl culture project in 1972,

In Indian waters, six species of p e a rl oysters are fou nd , viz., Pinctada fucata (Gould), P. m argaritifera (Linnaeus), P. chemnitzii (Philippi), P.sugillata (R eeve), P.anom ioides (Reeve) and P. atropurpurea (Dunker), A lth o u g h several s p e c ie s of pearl o y s te rs occur in the sea, only a few

have been found to produce pearls of gem q u a lity. The two s p e c ie s P. fucata and P. m a ig a iilifo ra stan do ut distinct from o th e r species in th is respect. The

Indian p e a rl oyster, P. fucata occu rs in e xte nsive beds in the G u lf o f Mannar and to a le s s e r extent in th e G u lf of Kutch. T h e black-lip p e a rl oyster, P.

m a rga ritifera confined m o s tly to Andam an and Nicobar w a te rs are fished m ore for th e shells than fo r th e ir pearls.

In 1973, the C.M.F.R.I. had successfully developed a n indigenous technology fo r the production o f cultured pearls a n d made the breakthrough in the hatche ry production o f s e e d s o f the Indian pearl oyster, P .fucata In 1981. The com plete package o f technology m a d e the pearl cultu re Industry to flourish in India.

In In d ia n waters, (here a re two spaw nings o f pearl oyste rs in a year, one in Jun~Sep. and the other in Nov-Feb, coin cid in g with s o u th -w e s t and north-east m o nsoo n respectively. H ow ever in th e Tuticorin H a rb o u r farm , the inactive, m aturing and mature g o n a d s were fo u n d alm ost in all th e m onths of the year (C hellam , 1987).

N orm ally, the pearl o y s te rs with ripe g o n a d are not pre fe rre d for nucleus im plantation as the g a m e te s ooze ou t resulting in d isp la ce m e n t or rejection of "nu cleus" or "graft tis s u e " or both th ro u g h the incision path. High mortality has been reported in th e pearl oyster a t th e maturing a n d spawning season as in other bivalves (W a d a e/ a/., 1989). Hence the com m ercial pearl production is always in te rru p te d due to maturation. In th is condition the de velopm ent o f triploid ste rile pearl oyster cou ld overcome th is problem (Wada et al., 1989; Lin and Jia n g , 1993).

A d u lt triploids are u s u a lly sterile due to the inability o f hom ologous chrom osom es to synapse du ring m eiosis w hich re su lts in uneven o r aborted separation o f chroniosoine trip le ts. In such sterile animals, m e ta b o lic energy norm ally u tilize d for gonad d e velop m en t m ay b e expected to b e diverted into continued s o m a tic growth (S tan ley ef a(., 1981). Sterility is advantage ous in situations w h e re the control o f reproduction is desirable. T rip lo id bivalves exhibit re duce d gonadogenesis and gam etogenesis when com p a re d to their

diploid e q uivale nts and furthe r m ore, induced trip lo idy is a ls o expected to

enhance physiological activity such as d ise ase resistance (B e a u m o n t and Fairbrother, 1991).

In molluscs, c y to ch a la sin B ha s been used to induce polyploidy th o u g h its negative e ffe c ts on survival have often b e e n reported (Stanley e t a i , 1981; and T a b a rin i, 1984).

T h e rm a l shock, as it is a simple m ethod, is e m p lo ye d in the inducement o f polyploidy in bivalves.

In th e present study, h e a t shock and c o ld shock were g iv e n to the embryos o f th e Indian pearl o y s te r, Pinclada fu c a ta at their developm ental stages. Inducem ent of polyploidy in P. fucata has g o t trem endous v a lu e in pearl culture. A ny achievem ent in th e inducem ent of p o lyp lo id y and its p o sitive result would boost th e pearl culture ind ustry in the co u n try. The study m a y facilitate the availability o f sterile pearl o y s te rs for pearl pro d u ctio n th ro u g h o u t th e year,

T h e m a jo r objectives o f th e present s tu d y w e re :

i. To in ve stig a te the optimal procedures ie., s h o ckin g time and tem perature for the ind u ctio n of polyploidy.

ii. T o c o m p a re the e fficie n cy o f heat s h o ck and cold s h o ck on ploidy induction and

iii. T o p re p a re the m etaphase chrom osom e sp re a d s of the p o lyp lo id pearl oyster larvae.

REVIEW OF LITERATURE

2. REVIEW OF LITERATURE

Increasing interests in chrom osom al engine ering and induction of polyploidy in m aricuture have often be en reported a s genetic im provem ent tools fo r successful production (M oav, 1976; Purdom , 1983). P olyploidy is one genetic m anipu la tion like gyn oge nesis and an d ro g e n e sis that m ig h t be helpful In aquaculture (W /lkins, 1981). P olypfo idy Is the c o n d itio n of p o sse ssin g more than twice the haploid number o f chrom osom es in th e cell nucleus. A triploid animal possesses three rather th a n two copies o f each ch ro m o so m e and tetraploid having fo u r copies in th e cell nucleus.

The chrom osom e m a n ip u la tio n becom es fe a sib le during th e nuclear cycles of cell division and b a sica lly com prises th e addition or su b tra c tio n of a complete haploid o r diploid set in m eiosis or m ito s is respectively (Purdom, 1983). This m a y be achieved b y treating the re c e n tly fertilized e g g s either chemically w ith cytochalasin B (C B ) o r 6-Dimethyl A m in o Purine (6 -D M A P ) or physically by a p p lyin g thermal (h e a t o r cold) sho ck o r high p re s s u re shock.

Both the th e rm a l and pressure s h o c k s disrupt th e form ation of th e m etaphase spindles of th e developing e g g s and preventing replicated ch ro m o so m e sets from sep ara ting into polar b o d ie s at meiosis. B ut, C B inhibits m icro-filam ent formation in cells (M aclean-Fletcher and P ollard, 1980). T h u s it allows karyokinesis b u t prevents cyto kin e sis which results in a triploid z y g o te with a diploid fem ale pronucleus and su b se q u e n t syn g a m y w ith a norma! h a p lo id male pronucleus.

It is no t easy to a rre st the first m eiosis In most fishes, since this division ta k e s p la c e In the o va ry itself. The ploidy manipulation in crustacean

sheIJfish is a ls o difficult, b e c a u s e they b ro o d their eggs a n d embryos.

Similarly, th e European fla t oysters, Oslrea e d u lis are even le ss amenable to ploidy manipulation.

In most vertebrates, eggs usually m ature after com pletion of meiosis I w he rea s in in ve rte b ra te s m ature-eggs are arrested a t prophase of meiosis I a n d only after fe rtiliz a tio n or activation, th e eggs co m p le te meiosis I and II re le a sin g two polar b o d ie s. Thus the delayed m eiosis in eggs o f the Indian pe arl oyster, P inctad a fucata p rovide s a unique oppurtunity for ploidy m anipu la tion by a rre stin g both polar b o d y I and II.

2.1. In d u c e m e n t o f p o lyp lo id y

P olyploidy in a q u a cu ltu re w/as atte m p te d first in the carp, Cyprinus ca rp io by Makino a n d O jim a (1943) a n d w as followed b y studies on many o th e r fishes. The trip lo id y induction h a s been investigated for the molluscan aquaculture in A m e ric a to market th e American oyste r, Crassostrea virginica (G m e lin ) throughout th e year (Stanley e f a /.,1981).

Ind uctio n of p o ly p lo id y has a lre a d y been reported in many com m ercial bivalve m o llu scs by several a u th o rs (Stanley e t al., 1981 T abarini,1984; Chaiton and A lle n ,1985; D ow ning e ( a/.,1985; A lle n e t a/.,1986 Y am am oto and Sugaw ara,1988; Uchimura e t a /.,1989; W a d a e t a/.,1989 G endreau and Grizel.1990 and Nell et a!.. 1994). In Ind ia, research on polyploidy induction is lim ite d to only in fis h e s . Though, Ind ia has vast resources o f shellfishes, no p lo id y work has b e e n carried out in m olluscs.

2.1.1. A m e rican oyster

T h e first attempt to in d u ce triploidy in a bivalve mollusc. Crassostrea virginica w a s performed w ith C B by S tanley efa/.(1981).

S tan le y e fa /. (1984) observed th a t heterozygosity w a s higher in the meiosis I triploids produced by CB tre a tm e n t during the firs t 15 min after fertilization.

T h e optim al con centratio n of CB in inducing triploid production in C.Wrg//»cn w a s found to be 0 .2 5 m g/l for 10-15 m in (Barber ef a /.,1992). Allen and Bushek (1992) induced trip lo id y by inhibiting second p o la r body with Im g CB/I fro m stripped gam ete s o f C. virginica.

S ca rp a et a/. (1995) com pared the e fficie n cy of 6 -D M A P and CB in inducing trip lo id y in the e a stern oyster, C .virg in ica and they fo u n d that the efficiency w a s m ore in CB tre atm e nt.

T h e effectiveness o f cytochalasin B o n the ploidy a n d survival of D-stage la rva e o f C .virg in ica w a s studied b y S upan a/.(1996). Anderson and W allace (1996) Induced triploidy in unconditioned C. virginica, using nitrous o xid e at 7atm pressure fo r 15 min. and th e success rate w a s found to be 56.8% b y them .

2.1.2. P a c ific oyster

T h e low er tem perature between 18*’ and 20“ C w a s fo u n d to be effective in inducing triploidy in Crassostrea g ig a s when CB w a s used as an inducing ag e n t (Downing efa /.,1985).

C ha iton and Allen (1 9 8 5 ) induced trip lo id y In C. gigas, b y means of pressure tre a tm e n ts which a d m in iste re d at 60 00-800 0 psi for 10 m in duration at 10 m in a fte r fertilization and it re su lte d 57% trip lo id y. They also developed a technique to detect the ploidy le ve l b y flow cytom etry.

A lle n and Downing (1986, 1990) reported that th e partial reproductive ste rility observed in trip lo id oyster, C. gig as which g re a tly affected the re pro ductive physiology.

A p p lica tio n of therm al shock in inducing triploidy in C. g ig a s was studied by Q u ille t and Panelay (1 9 8 6 ). They o b serve d that the th e rm a l shocks, applied at 10 -15 min or 35-40 m in post- fertilization at 35 and 3 8 “ C fo r 10 min yielded upto 25 and 45% trip lo id em bryos respectively. They a ls o obtained 60% triploid embryos w he n th e treatment laste d for 20 m in a t same temperature.

D ow ning and Allen (1 9 8 7 ) produced 8 8 % triploidy in th e Pacific oyster, C. g ig a s w ith CB treatm ent. Effect of CB o n th e induction o f polyploidy in C. gigas has been studied b y G uo et a/.(1992). Shpigei e t al. (1991) investigated th e growth pattern, gam etogenesis, physiology and biochem ical com position o f one year old d ip lo id and triploid C. gigas.

T h e highest percentage(90% ) of trip lo idy w as obtained in C. gigas by Desrosiers e t al. (1993) by tre a tin g the fertilized e g gs with 300 \iM 6-DM AP at 15min after fertilization for 20 m in.

G e ra rd et a/. (1994a) attempted trip lo id y induction in C.g/gas with 6-DM AP and obtained a mean o f 85% triploids.

G uo e t a/.(1994) p ro d u ce d tetraploidy in C .gigas with h e a t shock by blocking m ito sis 1. They also p ro d u c e d upto 30% tetraploids by zygote-zygote fusion and plastom ere fusion u sin g polyethylene g lyco l (PEG).

G uo and A llen(1994) developed a unique procedure fo r the production o f tetraploid C. g ig a s b y inhibiting firs t p o la r body in e g gs from triploids w hich w a s fertilized w ith n o rm a l haploid sperm .

K om a ru e t al. (1994) studied the ultrastructure o f sperm atozoa from induced trip lo id C. gigas th a t triploid sperm resem bled diploid sperm and were significantly larger than th a t o f diploid sperm.

A il'trip lo id C. gigas w e re successfully p ro d u ce d by m ating tetraploids with normal d ip lo id s (Guo e^o/.,1 996 ).

2.1.3. S y d n e y r o c k o y s te r

Nell e t al. (1994) stu d ie d the farm ing potential of trip lo id Sydney rock oyster, S accostrea co m m ercia lis that triploid oysters could re a c h market size 6-18 m o n th s faster and m aintained h ig h e r drym eat w eigh t a n d higher condition ind ex. Cox et al. (1 9 9 6 ) studied the g o n a d developm ent in diploid and triploid S. commercialis.

T h e appllcalion o f C B fo r triploidy induction in S. com m ercialis resulted in g re a te r survival a n d triploid percen tag e than 6 -D M A P treatment (Nell et a/..1996).

S h o u b a i et al. (1994) produced tetraploid Jinjiang oyste r, C. rivularis by heat s h o c k , cold shock, chlorprom azinum and tra d itio n a l chineae

medicine and th e y found th a t traditional Chinese medicine yie ld e d highest percentages.

2.1.4. B ro od in g oyster

T h e plo id y m anipulation in brooding o y s te r is diffcult. However, Gendreau a n d G rize l (1990) in d u c e d triploidy a n d tetraploidy in E uropean flat oyster, O strea e d u lis with CB a n d th e y obtained p o s itiv e results.

In O s tre a rivularis, te tra p lo id y was in d u c e d by cold s h o c k applied 3 min b e fo re the first cle a va g e gave 30 and 28% respectively (Shoubai e t a /.,1992).

Yu (1994) produced trip lo id Ostrea g ig a s with cold s h o c k and further he ob served that the la rva e from tre a te d groups had an obvious growth increase compared with co n tro l.

C B w as found to b e effective in inducing trip lo id y in Osfrea cucullata a n d gave higher p e rce n ta g e o f trip lo id y than th e rm a l shocks (Zeng el a/.,1994).

H a w k in s et al. (1 994 ) studied the g e n e tic and m e ta b o lic basis In meiosis I trip lo id Ostrea edulis tre a te d with CB and they found th a t total dry tissue w e ig h t o f meiosis ) trlp lo id s w as more th a n diploid siblings o r meiosis II triploids d u e to increased he tero zygo sity In m e io sis I triplolds.

2.1.5. S c a llo p

T a b a rin i (1984) in d u ce d triploidy in th e bay scallop, Argopecten irradians w ith CB and stu d ie d the effect o f triploidy o n grow th and g a m etogenesis and he found th a t the adductor m u scle weight a n d total body

tissue (wet) w e ig h t in triploid s ca llo p s were g re a te r than diploid controls. He further ob served that the m ajority o f triploid scallops faile d to mature.

T h e m o st effective C B d o s e to induce trip lo id y in P ecte n maximus was 0.5 m g/l a t 10min after fe rtiliz a tio n for 20m in w h ich yielded 3 0 % triploid embryos (B e a u m o n t,1986). He a ls o suggested th a t polar body c o u n ts were the simple m ethod fo r assessing th e d e g re e of triploidy in embryos.

K o m a ru e f • a/. (1988) produced triploid scallops, C h la m ys nobilis with CB and detected the p lo id y levels by m icrofluorom etry.

T rip lo id black scallops, Chlamys vaha w e re produced by treating with CB (Baron e t a/.,1989). T h e y also found th a t there was n o significant difference in grow th between d ip lo id controls and triploids, but m o rta lity was higher for the triploids, before m etam orphosis.

C a n e llo ct a/. (1 9 9 2 ) induced trip lo idy in th e scallop, Argopecten p u rp u ra tu s with h e a t sho ck and they analysed the post-fertilization

time, tem perature and duration o f tine the rm a l shock for o b ta in in g highest percentages o f triploidy.

D e sro sie rs a/. (1 9 9 3 ) produced triploid giant s e a scallop, P/acopec/e/7 m agellanicus w ith 6-DM AP and th e y obtained 9 5 % triploid scallops. T h e y a ls o delerm ined th a t increasing th e treatm ent d u ra tio n improved the efficiency of triploid induction, b u t prolonged incubations with 6 -D M A P led to the d e velop m en t o f abnormal larva e.

W in k le r et al. (1 9 9 3 ) induced trip lo id y in A rgopecten purpuratus with CB a n d produced a m e a n proportion o f 17% triploids a n d they also

obtained 6 % triploids in the c o n tro l groups w h ic h were treated w ith 0,005%

Dimethyl s u lfo xid e (DMSO).

T h e heat shock of 3 1 “ C fo r 10 m in a p p lie d at 10 min p o s t-fe rtiliza tio n was found to be the optimal p ro c e d u re for trip lo id y induction in A . purpuratus (Toro e /a /.,1995).

Z e n g e /a /. (1995) fo u n d that the CB s h o ck was m ore effective than thermal s h o c k s in inducing trip lo id y in C hlam ys nobilis. They a ls o reported that the th e rm a l shock tre a tm e n t was an e ffe c tiv e and sim p le m ethod to induce trip lo id y from a com m ercia l point of view.

In Chlam ys nobilis trip lo id y was in d u ce d by treating th e fertilized eggs with C B and cold shock in order to arrest firs t polar b o dy and second polar body w h e re the retention o f second polar b o dy gave h ig h e r incidence of triploidy (Lin e f a/., 1995). He a ls o studied the g ro w th pattern of trip lo id scallop.

2.1.6. M u s s e l

Y a m a m o to and S u g a w a ra (1988) a p p lie d heat shock a n d cold shock for triploid in d uction in the mussel,/V/yW as eduds a n d obtained 9 7 .4 a n d 85.3%

triploidy re p e ctive ly. they also fo u n d that lo n g e r h e at shock tre a tm e n t(2 0 min duration) im p ro ve d the e fficie n cy o f triploid Induction.

B e a u m o n t and K e lly (1989) studied the larval g ro w th o f triploid MyiiJus e d u lis which were p ro d u c e d by heat sho ck and CB treatm ent.

Electrofusion te ch n iq u e w as proven to induce polyploidy in Mytilus edulis a n d M. galloprovic/al/s u p to 36% trip lo id s and 26% te tra p lo id s when

11

electric p u lse s were applied p rio r to com pletion of the firs t cell division (Cadoret, 1992).

S carpa e t a!. (1 9 9 3 ) reported th a t tetraploids and pentaploids were produced in M. galloprovicialis by inhibiting b o th polar b o dy I and II with a continuous C B treatment fro m 7 -3 5 min after fertilization.

D e sro sie rs ef a/. (1 9 9 3 ) produced triploid M. edu//s with 6 -D M A P and they found th a t the normal d e ve lo p m e n t in tre a te d groups was lo w and the percentages o f abnormal la rva e w e re related to th e duration of tre a tm e n ts.

Six different shocks like CB (1mg/l), h e a t (30° C), c a lc iu m (0.1/W), caffeine (15m M ), combined ca lciu m -h e a t ( 0 .1 a t 3 0 ” C) and com bined caffeine- h e a t(1 5 m M a t 3 0 “ C) shocks w e re compared in inducing trip lo id y in Mytilus galloprovincialis by Scarpa e t a /.,1994. They h a v e reported that C B was the most effective in producing v ia b le triploid individuals.

In M. chilensis, trip lo id y w a s induced w ith heat shock w h ic h yielded 51% triploid em bryos and th e triploid larvae show ed diffe ren ce in shell length than th a t o f control (T o ro and Sastre, 1995).

2.1.7. C lam

A lle n e t al. (1986) p ro d u ce d triploid s o ft-s h e ll clam, M ya a rena ria with CB tre atm e nt and studied th e gam etogenesis a n d sex ratio o f th e treated groups.

T h e chemical shock tre atm e nt of 0.5m g CB/1 applied e ith e r from 0-15 min o r fro iT i 15-30 min after fertilization gave th e best results in triploidy

12

induction in the Manila cla m , Tapes sem idecussatus (B e a u m o n t and Contaris, 1988),

M ason e t a/. (1988a & b) reported that th e triploid Mya a re n a ria would grow larger and faster than th e ir d ip lo id siblings d u e to retarded gam etogenesis and more heterozygous in trip lo id s . In contrast, the triploid Mercenaria mercenaiia tre a te d with CB sh o w e d sm aller d ry tissue w eight a n d all shell parameters th a n diploid controls o v e r three grow th seasons (Hidu e t a /.,1988).

G o slin g and Nolan (1 9 8 9 ) proved that th e rm a l shock o f 3 2 °C for 10 min. yielded b e tte r results tha n C B treatment in triploidy induction In Tapes semidecussatus.

T h e effective CB tre a tm e n t for the in d uction o f triploidy in Ruditapes philippinaw m w a s developed b y Dufy and D ite r (1990). In th e sam e year, Diter and D u fy (1990) induced tetraploidy in em b ryo s of R. p h ilip pina rum by treating the e g gs with CB a t 5 and 45 min a fte r insemination. T h e y also reported th a t no tetraploids w e re detected in 4 m o n th -o ld spat.

U ttin g and Doyou (1 992 ) studied th e utilization o f egg lipid reserves fo llo w in g induction o f triploidy in Tapes philippinarum and they found that th e percentage o f trip lo id s was h ig h e r fo r the em bryo s fro m brood stock fed on Ske/eJonema th a n fro m fed on D unaliella.

G e ra rd et al. (1 994 b) obtained 95% trip lo id embryos in Ruditapes decussatus w ith 1 mg CB/1 a p p lie d at 15 m in a fte r fertilization fo r 20 min. In

T. ph ilip pina rum application o f 0 .5 m g CB/I yie ld e d 70-77% trip lo id with 45%

mean su rviva l to (he D -stage la rva e (Utting a n d Child, 1994).

13

T rip lo id y was induced in fertilized e g g s o f Tapes phW ppinarum with CB at m e io sis \l. The growth rate, survival, fo o d consumption, respiration rate and b io ch e m ica l content o f diploid and trip lo id larvae w e re studied for comparison a n d found to be sim ila r {Laing and U tting, 1994).

Nell e t at. (1995) in d u ce d triploidy in T. dorsatus with 1 m g CB/1 for 15min exposure and further they observed that th e re w as no d iffe re n c e in the growth rate o f d ip lo id and triploid larvae.

2.1.8. P earl o y s te r

In d u ctio n o f polyploidy ha s also been reported in pe arl oysters by various authors. Uchimura et at. (1 9 8 9 ) induced trip lo id y in the J a p a n e s e pearl oyster, Pinctada fucata m a rte n sii by inhibiting firs t polar body w ith 0.5mg CB/I treatment. T h e pioidy le v e l w a s estimated b y m icrofluorom etry w ith 4',6- diamidino-2 phenyiindole (DAPI) staining and fo u n d to be 65.4% trip lo id y.

W a d a e t at. (1989) p ro d u c e d 100% trip lo id y in P. fucata m a rie n s ii by inhibiting se co n d polar body w ith C B treatm ent and they fo u n d th a t cold shock(6.5° C ) w a s also effective w h ic h gave 52% trploidy in the J a p a n e s e pearl oyster.

T h e effect of com bination of caffeine and heat shock treatm ent In inducing trip lo id y in P. fu ca ta m a /ie n sii w as investigated by D u ra n d et al.

(1990).

S h e n e t a/,(1993) fo u n d that a h yd rosta tic pressure o f 2 0 0 -2 5 0 kg/cm at 5-7 m in a n d 17-19 min post-fertilization for 10 m in was m o st effective to induce trip lo id y in P. m artensii w h ich gave 76% triploids with high hatchability.

14

Jian g e t a!. (1993) p ro d u c e d triploid P. m a riensii with C B treatm ent and studied th e grow th of triploid p e a rl oyster. T h e y observed that th e growth rate of a d u lt triploids w e re significantly h ig h e r than diploid siblings especially d u rin g reproductive seasons. They a ls o suggested th a t th e faster growth rate o f triploid adults is caused by th e retarded d e ve lo p m e n t of gonads in trip lo ids.

Lin e t al. (1996) o b serve d th a t the m o rta lity in the triploid P.m artensii was higher d u rin g the developm ental stages fro m D-stage to ju v e n ile but, there was no difference in m o rta lity between d ip lo id s and triploids during the stages of em b ryo s and adults.

He e t al. (1996) s tu d ie d the gam etogenesis, fe rtiliz a tio n and development o f the triploid P. m a tie n s ii and th e y concluded th a t th e triploid pearl oyters w e re sterile.

Lin e t al. (1993) produ ced perfect pearls fro m triploid P .m a rte n s ii with higher returns tha n from diploid p e a rl oysters. T h e y also suggested th a t the induced triploid pearl oysters w o u ld be useful fo r shortening th e period of pearl culture and producing g o o d quality pearls.

15

P late 1. The Indian pearl oyster, Pinctada fucata (G.) brood stock collected from natural pearl beds, Tuticorin.

D E S C R IP T IO N OF S P E C IE S

O R D E R F A M IL Y G E N U S S P E C IE S

D ysodonta P te riid a e P in cta d a fu c a ta (Gould)

C o m m o n name : In d ia n pearl oyster.

Distinct c h a ra c te rs

T h e hinge of oyster is fa irly long and its ratio to the b ro a d e s t width of the shell is about 0.85 and th a t to the d o rso ve n tra l m easurem ent is about 0.76. The le ft valve is deeper th a n the right. H in g e teeth are p re s e n t in both valves, one each at the a n te rio r and posterior ends of the lig a m e n t. The anterior e a r is larger than In th e o th er species a n d the byssal n o tch , at the junction o f th e b o d y of the shell a n d the ear, is slit-like . The p o sterio r e a r is fairly well developed. T h e outer su rfa ce o f the shell va lve s is reddish o r yellowish- brown w ith radiating rays o f lighter colour. T h e nacreous la y e r is thick and has a bright golden-yellow m e ta llic lustre.

Biology

P e a rl oysters are s e d e n ta ry animals fo u n d attached on d e a d corals, rocks and also on sand grits by means of b y s s u s threads. T h e y are filter feeders. T h e y m ainly feed on phytoplankters. T h e y Inhabit at d e p th s from 10 to 20 metres.

In pearl oysters, th e sexes are sep ara te and they d o no t exhibit sexual d im o rp h ism . P. fucata m ature when th e y a re 8 m onths o ld and about

16

17-25 mm size. They spawn in th e ninth m onth coisiding with th e monsoon seasons.

T h e longevity of a p e a rl o yste r is about 5 to 6 years and th e maxim um attainable size is 100 min.

Distribution in India

P. fu c a ta are found in la rg e numbers on "P a a rs" in the G u if o f Mannar

which extend fro m Kilakarai to Kanyakum ari and le s s e r extent In th e Gulf of Kutch on in te rtid a l reefs kno w n as "Khaddas". In Palk Bay th e p e a rl oysters are found on coarse sandy b o ttom .

C ollection

P earl oysters are co lle c te d by skin d iv in g and S C U B A diving.

17

MATERIALS AND METHODS

3. MATERIALS AND METHODS

3.1. Materials

3.1.1. Spawning, Fertilization a n d In d u c e m e n t o f p o ly p lo id y 3.1.1.1. C ollection o f oysters

Sam ples of Indian p e a rl oyster, P inctada fucata were collected from the natural p e a rl banks in th e G u lf of Mannar. Tuticorin located a t 08°

47'N ; 78° 08'E. T h e oysters with rip e gonad were sele cted and c le a n e d to rem ove fouling organism s. Since th e experim ents w ere conducted in the natural spawning season, no co n d itio n in g was done p rio r to spawning.

3.1.1.2. M aterials a n d Equipm ents a. Knife to rem ove fouling organism s.

b. Perspex ta n k (30 litre capacity) c. Therm om eter

d . Jum otherm om eter e. Immersion h e a te r f. Electronic therm ostat g. 30/jm and 100/.jm sieves

h. Light m icro scope and em bryo cup,

i. Beakers fo r g a m ete handling a n d larval rearing.

3.1.1.3. C hem icals

a) Neutral b u ffered formalin (4 % )

b) Ethelene d ia m in e tetra acetic a cid (EDTA) for se a w a te r treatm ent.

18

3.1.2. Ploidy verification

3.1.2.1. M aterials a n d Equipm ents a) A pair o f droppers

b) Test tubes and pipettes to p re p a re solutions.

c) Screw ca p p e d vials of 5 ml c a p a c ity to store fix e d embryos.

d) Insulated b o x and ice cubes fo r storage of fixed em bryos.

e) Clean slides.

f) Slide w arm er

g) Hot plate w ith magnetic stirrer.

h) Coplin ja r fo r staining, i) Filter papers.

j) Slide storing cabinet

.k) Nikon M icro sco p e with photom icrographic attachm ent.

3.1.2.2. C hem icals

a. Colchicine pow ders and so lu tio n s o f strengths 0 .0 2 % for arresting o f m itotic metaphase chromosomes.

b. Seawater a n d distilled water as hypotonic solution.

c. Glacial a c e tic acid and m ethanol to prepare C a rn o y ’s fixative.

d. 50% A ce tic acid.

e. Giemsa s ta in stock solution.

f. Di-sodium hydrogen phosphate and potassium di-hydrogen p h o sp h a te to prepare S o re n s o n ’s phosphate buffer (pH 7.0).

19

3.2. Methods

3.2.1. S paw ning a n d fertilization

Spawning in pearl o y s te rs was induced by thermal stim ulation.

The oysters w e re placed in 30 litre s of filtered s e a w a te r taken in a perspex tank with (he size 50cm x 30cm x 3 0 cm. The w a te r tem perature w a s raised from the am bient tem perature o f 2 8 to 30“ C using an immersion he ate r. The required tem perature was set w ith the help of Jum otherm om eter. A e ra tio n was provided during the process in o rd e r to disperse th e h e a t uniformly in th e tank.

T h e immersion heater and Jum otherm om eter were connected to an autom atic thermostat.

If the oysters were n o t spawned at 3 0 “ C, the water tem perature w a s further raised to 32° C. D u rin g the process o f gradual h e a tin g , the oysters were ind uced to spawn. O n c e the oysters sta rte d spawning, th e y were individually isolated in a s e p a ra te beaker a n d allowed to c o n tin u e the

spawning till th e gametes were re le a se d fully.

The eggs o f three or fo u r fem ales were p o o le d together a n d sieved through 100 /jm nylebolt cloth. L a rg e dust, faecal m a tte rs and broken pieces of byssus threads w e re retained in th e cloth. The e g g s alone passed th ro u g h the mesh. The e g gs thus collected w e re kept for fe rtiliza tio n in 1 litre o f filtered seawater.

S im ilarly, the sperm w e re a lso collected b y passing th ro u g h 30 pm sieve. Eggs a n d sperm were k e p t separately at 2 4 ± 1 " C before fertiliza tion to synchronize th e egg development.

20

T h e tim e, when the spe rm were a d d e d to the s u sp e n sio n of eggs was considered to be the start o f the experiment. T h e eggs and sperm were gently mixed fo r proper fertiliza tion . After five minutes, the eggs-sperm suspension w a s made upto 5 litre w ith isothermal filtered seaw ater and divided into ten a liqu ots o f 500 ml each. T h e embryos w e re maintained a t th e ambient tem perature o f 24±1'’ C till they w e re given the rm a l shock,

3.2.2. In d u c e m e n t o f polyploidy

E m b ryo s (SxlO**) o f e a ch aliquot were ke p t in a PVC p ip e (6cm dia;

9cm hight) fixe d w ith 3 0 /jm sie ve cloth at one end. The embryos w e re rinsed with filtered se a w a te r to rem ove excess sperm. Inducement o f po lyp lo id y was done by im m e rsing the sieve containing the em bryos in heated o r chilled seawater.

3.2.2.1. E x p e rim e n t 1: e ffe c t o f timing o f initiation o n poiyploidy in du c em ent

T o investigate the e ffe c t of tim ing o f initiation on polyploidy inducem ent, e a ch aliquot was tre a te d one by one at 30° C at 10, 15, 2 0 ,.,.and so on upto 5 0 m ill after fe itiliza tio n w ith an interval o f 5 min. T h e duration of each treatm ent lasted for 10 m in. T h e remaining o n e was untreated and kept as control. A t e v e ry 5 min, of po st-fe rtilization , a sam ple of em bryos w as taken from the con trol and fixed in 4 % neutral buffered form alin. Polar b o d y extrusion tim e was e stim a te d from the m icro scopic observations of the fix e d embryos.

3.2.2.2. E x p e rim e n t 2: effect o f h e a t shock on p o iy p lo id y in d u c e m e n t

B ased on the results obtained from Experiment-1, th re e different tem perature v a lu e s 32, 35 and 38 ±0.5° C w e re investigated. T h e embryos w ere divided in to fou r aliquots a n d treated at 10, 2 0 and 50 min post-fertilization

21

Plate 2. A view o f experimental setup for inducing polyploidy by heat shock.

u m .

Jiew embryos under heat shock treatment. (A close

Plate 4. Cold shock treatment of developing embryos by using ice.

P la te 5. Rearing o f la rv a e after th e rm a l shock tre a tm e n ts .

for 10 min duration in order to block m eiosis I, meiosis (J and first cleavage respectively. Remaining one was kept as control.

3.2.2.3 E x p e rim e n ts : effect o f c o ld shock on p o ly p lo id y in d u c e m e n t

Sim ilar to Experiment-2, the embryos w ere also treated at lower temperature o f S'* C ( ± rC ) at 10, 20 and 50 m in after fertjlization fo r 15 min duration. The untreated one was ke p t as control.

3.2.3. L a rv a l re a rin g

After treatment, em bryos were reared in 1 litre of filtered seawater, maintained at 23±1°C. Seawater for all treatments and larval rearing was filtered and treated with 1 mg/1 ethylene diamine tetra acetic acid (EDTA) as a precaution against metal contamination (Utting and Helm, 1985).

3.2.4. P loidy verification

Ploidy level of all treated groups as w ell as control w as determined by the partially modified direct chromosome counts methodology o f Klingerman and Bloom (1977). The trochophore stage was considered to be th e best stage for direct chrom osom e counts. I {etice,10 hrs after fertilization, a portion of free swimming larvae were collected from each group and treated with 0.02%

colchicine for 2 hrs. Then they w ere transferred to a hypotonic solution (1 part seawater+ 2 parts distilled w ater) for 30 min. Before fixation, Cornoy's fixative was added at 1:10 ratio to the hypotonic solution to prevent cells bursting upon sudden exposure to full-strength Cornoy's fixative.

Subsequently, the lan/ae were fixed with fresh Cornoy's fixative (3 absolute m ethanol; 1 glacial acetic acid) fo r 2 hrs at 4°C. During fixation, th e fixative was changed 3-4 times. Later, the larvae were taken out and excess fixative was removed as for ns [)os';iblo. 1 hn fixed Inrvno w o io dissocinlod in 50% ncotic

9?

acid immediately. The suspension o f larvae was pipetted out onto a clean, pre­

warmed (45‘’C) microsiides. Then the slides were air-dried and stained in 5%

Giemsa (5ml G iem sa stock + 95m l Sorenson's phosphate buffer) at pH 7.0 for 20 min.

The clear metaphase spreads w ere only counted. The percentage o f ploidy level o f each group was estimated by th e frequency distribution m ethod (Wada e t a/.,1989). For each treatment 25-35 metaphase spreads were counted.

The chromosome num ber (n=14, 2n=28) of P. fucata has already been reported and conformed by Alagarswami and Sreenivasan (1987). Hence, th e ploidy level was described as following: chrom osome counts in the range 26-28 = diploid(2n); 39-42= triploid(3n) and 52-56 = tetraploid(4n).

3.2.5. D ata a n a ly s is

The effect of thermal shock on polyploidy induction w as determined using analysis o f variance test and students' t-test analysis w e re adopted to test the differences between treatments.

23

RESULTS

4. RESULTS

4 .1. Spaw ning a n d fertilization

Pearl oysters were spaw ned at 34 ± 1° C and the response was noticed first in m ale oysters followed by females. T h e addition of stripped sperm to the spawning medium gave better spawning results. The fertilized embryos were also obtained by “stripped gametes" where the time of fertilization is controllable (Allen and Bushek, 1992). By following this method the percentages o f undeveloped embryos were found in higher numbers. Hence, to obtain gametes, therm al stimulation w as carried out in all the experiments and the fertilization rate w a s recorded as m ore than 95%.

4 .2 . E xp e rim e n t 1: effect o f tim in g o f initiation o n polyploidy in ducem ent b y heat s h o c k

The fixed embryos were observed for polar body extrusion under light microscope (20x). Most of the eggs completed the first meiosis at 15 minutes after fertilization a t 2 4 ± r c . The second meiosis and first cleavage w ere found after 25 m inutes and 55 m inutes respectively after fertilization at 2 4 ± rC (Table 1).

The heat shock at 30°C was given to induce polyploidy. The percengage o f aneuploidy was observed in low percentages in all treated groups. Triploidy was noticed In all the treated groups (Table 2) ranged from 10.3 ± 1 .4 5 to 31.0 ±2.08% . In the control groups triploids and tetraploids w ere not found. The highest percentage of triploidy was obtained in treatment group 1 and 3 w hich was corresponding to extrusion of first and second polar bodies. The percentage of triploidy (31%) in the treatm ent group 3 (20 minutes after fertilization) w as significantly higher (F(gjo)= 13.9736, P< 0.01) than that of

24

P late 6. M etaphase chrom osom e spreads from d ip lo id em bryos of P. fu ca ta

Plate 7 . Metaphase chromosome spreads form th p h id embryos o f P. fucata

P late 8 . M etaphase c h ro m o s o m e s p re a d s form te tra p lo id em bryos ot P . fu ca ta

T a b le 1. The Percentages o f u n treated eggs w ith po lar body I (P B I), polar b o d y II (P B II) and 2’ Cell stage (E xperim ent 1).

Time after Fertilization

(min)

PBI {%)

PB II (%}

2 - cell stage {%)

Total Number of eggs observed

5 OtO.OO 50

10 25+1.15 ^{- -} 75

15 64±173 0+0.00 - 70

20 73+3.51 26.3±3,28 ^- 69

25 - 71.0±1.15 - 90

30 - 81.0±0,57 - 53

35 ^- 85.6±1.76 - 47

40 - 83.6±2.33 - 50

45 - - 0+0.00 65

50 * - 18.6±2.33 74

55 - - 52.0+1.53 68

60 ^• • 70.3±1.45 70

Values are m ean ± S E (n=3)

25

-A -2 -C e ll S ta g e

Tim e a fte r fertilization {m in.)

Fig. 1. T im ing of PB I and PB II extrusions and 2>cell sta g e from fertilized eggs in exp erim en t 1

26

T a b le 2. E ffe c t o f in itia tio n tim e on p o ly p lo id y in d u c e m e n t a t 30®C (E x p e rim e n t 1).

Treatm ent groups

Initiation time o f Treatment

(min)

Dtploidy {%)

Triploidy (%)

Tetraploidy {%)

1 10 74.0±2.64 29.3±2.18

2 15 86,0±1.53 14.0+1.52 ^-

3 20 68.0±1.73 31.0±2.08 1.0+0.57

4 25 82.0±2.88 15.3±1.45 2.6±1.45

5 30 76.6±1.45 18.6±1.45 4.6±1.45

6 35 79.3±2.73 14.3±1.45 6.3±1.76

7 40 78.6+2.40 11.3±1,85 10.0±0.58

8 45 76,0±1.53 10.3±1,45 13.6±0.88

9 50 60.3+3.93 18.3±3.53 21.3±1.85

10 Control 100.0t0.00 ^{“ •}

V a lu e s are m ean ± SE (n=3)

27

T a b le 3. A N O V A Table s h o w in g th e s ig n ific a n t va ria tio n b e tw e e n the in itia t io n tim e a n d t r ip lo id y p e rc e n ta g e s

Source of

variation ^{d f} SS M S F

Treatment ® 8 1333.1851 166.6481

F=13.9736**

Error 18 214.6667 11.9259

T o ta l 26 1547.8518

- Time.

- Significance at 1% level

T a b le 4. A N O V A ta b le s h o w in g th e s ig n ific a n t d iffe re n c e b e tw e e n in itia tio n tim e a n d in d u c e d te t r a p lo id y p e rc e n ta g e

Source of Variation df SS MS F

Treatment ^ Error

6 14

909.9047 73.3333

151.6507

5.2381 F=28.95’ *

Total 20 983.2380

- Time.

** - Significance a t 1% level

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all other treatments (Table 3) except the treatment group 1 (10 m inutes after fertilization, 29.3% triploids). There was no significant difference (t = 0.55, p>0.05) between treatment groups one and three.

The highest percentage (21.3+ 1.85%) o f tetraploidy w a s obtained from treatment group 9 (50 m inutes after fertilization), which w a s highly significant (F(6,i4) = 28.95, P< 0.01) than other treatm ent groups (Table 4).

4 .3 . E xp e rim e n t 2 a n d 3: effect o f h e a t shock a n d c o ld shock o n polyploidy inducem ent.

Five temperature shock treatments w ere employed fo r producing triploidy and tetraploidy (6,30,32,35 and 38®C). The highest percentage of triploidy (74.3 ± 2.33%) was found at 35°C treatm ent group w hich was significantly higher (F = 5.2118, p<0,05) than other treatment groups except 38°C (64.6%). The triploidy percentages 31.0%, 37.0 % and 49.3% at SOX, 32°C and G^C w ere noticed respectively (Table 6). There was no significant difference (P>0.05) between groups treated at 35 and 3 8 X . Despite insignificant difference observed between the triploidy at 38°C treated groups and 35°C, abnormal larvae were observed in higher numbers in 38°C group.

The highest percentage o f tetraploidy (46.6% ) was noticed (Table 6) at 35°C temperature which was significantly higher (F (

4

^,

10

) = 17.7883, P<0.01) than other treatment groups (Table 7) except the groups treated at 6 and 38°C. The abnormal larval percentage was higher in tetraploidy groups treated at 3 8 X like triploids.

30

Table 5. Percentages o fd ip lo id y , triploidy a n d tetraploidy in d u c e d at d ifferen t temperatures.

T reatm ent Tem perature

( C )

Initiation Time

of Treatment

(minj

Duration

i

D ip lo id y

(%) ^{T riploidy}(%)

Tetraploidy

(%)

ia 6

10 20 50 C

15 15 15

50.6±3.48 61.6±2.03 45.0±2,64 100±0.00

49.3±3.48 37.6±2.02 14.3±0.88

1.0±0,57 40.6±3.38

2 '•b 30

10 20 50 C

10 10 10

74.0±2.64 68.0+1.73 60.3±3,93 100.0+0.00

26.0+2.64 31.0±2.08 18.3+3.52

1.0±0.58 21.3±1.85

3. 32

10 20 50 C

10 10 10

69,0±2.08 63.0±2.31 50.3+4.37 100.0+0-00

31.0±2.08 37,0±2.31

18.3±2.02 31.3±2.73

4. 35

10 20 50 C

10 10 10

35,6±2.96 21.3±3.84 32.6+1.73 100±0,00

64.3±2.96 74.3±2.33 20.6±2.40

4.3±1.76 46.6±1,66

5. 38

10 20 50 C

10 10 10

36.0±2.08 25.0±2.51 28.3±4.41 100±0.00

64,0±2.08 64.6±2.60 27.6+3.38

10.3±0.88 44.0±2.31

Values are m ean + SE (n-3)

® - data obtained from experiment 3

^ - data obtained from experiment 1

31

D 1 0 m in.

0 2 0 m in B 5 0 m in

□ C ontrol

T e m p e ra tu re (°C)

Fig. 3. E ffe c t of thermal s h o c k on trip lo id y induction at d iffe re n t treatm ent times

32

B IO roVn

020 n^'n H50

E3 Control

T e m p e ra tu re (°C)

Ftg. 4. E ffe c t o f tlio rm al s h o c k on te tra p lo ld y induction a t d iffe re n t tre a tm e n t tim es

33

T a b le 6. A N O V A table s h o w in g th e e ffe c t o f te m p e ra tu re in in d u c in g t r ip lo id y a t 10,20 a n d 5 0 m in . a fte r fe r tiliz a tio n

Source of variation

df SS MSS F

^Sample 2 2637.504 1318.752 F =13.2130**

^Treatment 2 4 2080.716 520.179 F = 5.2118*

Error 8 798.456 99.807

Total 14 5516.676

1

2

*1r

*

Time o f initiation of Treatm ent after fertilization Tem perature of the treatm ent

Significance at 1% level Significance at 5% level

Table 7. A N O V A table s h o w in g th e e ffe c t o f te m p e ra tu re in in d u c in g te tr a p lo id y a t 50 m in a ft e r fe rtiliz a tio n

Sourcc of variation

df SS MS F

Treatment’ . Error

4 10

1299.7333 182.6666

324.9333 18.2666

F = 17.7883**

Total 14 1482.3999

’ - Tem perature of the treatm ent

*• - Highly significant at 1% level

34

4 .4 . C om parison o f the e ffe c t o f h e a t shock a n d co ld shock fo r triploidy an d te tra p lo id y inducem ent.

From the results obtained (Experiment 2 and 3) the overall triploidy percentage w as comparatively lo w e r in cold shock than that of heat shock at 35 a n d 38"C and liig h e r in cold shock than heal shock at 30 and 32'’C treated g ro u p s .

U nlike triploids in tetraploidy there w as no significant difference ( t = 1.59, P>0.05) between 6 and groups treated at 50 min after fertilization.

T rip lo id y percentage obtained from 35°C groups treated at 10 and 20 min after fe rtiliza tio n w ere highly significant (t=6.161, P<0.05) than triploids produced by c o ld shock at th e same time treatm ent. For tetraploidy percentage, there was no significant difference (t=1.59, P >0.05) between 35 and 6 “C groups treated at 50 m in after fertilization.

DISCUSSION