Aquaculture Research. 1998. 29. 77S-7Si

Blocking polar body with cytochalasin B in the

fertilized eggs of the small abalone, Haliotis

diversicolor supertexta (Lischke), and the

development and ploidy of the resultant embryos

H-S Yang'^, Y-Y Ting"' & H-C Chen''Department of Zoology. National Taiwan University, Taipd. Taiwan. ROC

^Tainan h'ish Culture Station ol" the Taiwan Fisheries Research Institute. Keelung, Taiwan. ROC

Correspondence: Hniig-Shii Yang, #4. Hai-Phu. SHII-KU. L'hi-Ku. 'lainan. Taiwati. ROC

Abstract

The efFecls of blocking polar body I (FBI) orpolar body II (PB2) with four different dosages ofcytochalasin B (CB) on the development andploidy of resultant embryos were studied in thesmall abalone. HalioUs diversicolor supertexta(Uschke). To block the release of PBl. thefertilized eggs were treated with 0.25. 0.5. 1.0or 2.0mgL~^ of CB for 10 min beginning at3 min post-fertilization at 24 °C. To block therelease of rB2. lhe fertilized eggs were treatedunder the same conditions as PBl. except thatthe treatment was begun 10 min post-fertilization.In the control group, only 41.8% of the cellshad a diploid number of J2 chromosomes.although spontaneous haploids (9.0%). tripolids(7.5'^) and aneuploids 141.7'^) were also ob-served. In CB treatment of PBl and PB2 groups.5.0-28.6% of the cells remained as diploid.triploids (IO.O-IH.9%) and aneuploids (41.1-(•}l.0%]. With regard to the development ofthe resultant embryos, the proportion ofnormal embryos in the control group was87%. while in the treatment groups, the propor-tions of normal embryos in the PBl and PB2groups were 57-58% and 53-56% in the0.25 mg L"* and 0.5 mg L"' CB treatments, re-spectively. From this data on induced triploidsand the resultant development of normalembryos, the proportions suggest that 0.25-().5mgL~' of CB for lOmin was sufficient for

blocking the release of PBl or PB2 to produce

triploids in the small abalone.

Introduction

The potential of the chromosome manipulationtechnique with clouding temperature (Quillet &Panelay 1986: Fujino. Okumura & Inayoshi 1987).pressure (Chaiton & Allen I9HS; Aral. Naito &Fujino 1986) and chemical treatments (Stanley.Allen & Hidu 1981: Tabarini 1984; Beaumont &Contaris 1988| to induce triploidy or tetrapioidy inorganisms has previously been discussed. In mostvertebrates, eggs mature after completion of meiosisI. but in many marine mollusks. mature eggs arearrested at prophase I of meiosis I (Ahmed 1973; Lu1986: Strathmann 1987). These eggs only completemeiosis I (i.e. release polar body I. PBl) and meiosisII (i.e. release polar body II. PB2) after fertilization oractivation. The interruption of meiosis in eggsprovides a unique opportunity for the manipulationof PB i or PB2 tripioids. Triploids are organisms withthree sets of chromosomes in each cell which cantheoretically cause sterility because homologouschromosomes cannot synapse in meiosis. This hasbeen well established in some species of lish(Benfey. Sutteriin & Thompson 1984: Chourrout1984) and molluscs (Longo 1972: Beaumont &Fairbrother 1991). Stanley etal (1981) usedcytochalasin B (CB) during PBl. which resulted intriploid and tetraploid embryos in the Americanoyster. Crassostrea virginica (Gmelin). Similarly, both

1998 Blackwell Science Ltd. 775

Cytochalasin B and small abalone eggs HS Yang et al. Aquacuhure Research. 1998. 29, 775-783

triploid and tetraploid embryos were produced byblocking PBl in the blue mussel. Miitilus edulis(Lamarck). (Yamamoto. Sugawara & Oshino 1988)and Pacific abalone, HaUotis discus hannai (Ino) (Araieta}. 1986). Stephens (1989) reported that CBblocking ol PBl in the Pacific oyster. Crassostreagigas (Thunberg), produced tetraploids. not triploids.Guo. Cooper. Hershberger & Chew (1992) reportedthat CB acting on the PBl of the Pacific oysterresulted in diploids. triploids. tetraploids and amajority of aneuploids in embryos. This metaboliteof a fungus prevents cytokinesis, but not chromo-some replication (Copeland 1974). Cytochalasin B iswidely used in the production of PB2 triploids in thePacific oyster, and it is an important part of theoyster culture industry in USA and Canada becauseof the sterility of these animals (Chew 1984: Allen.Downing & Chew 1989). Stanley. Hidu & Allen(1984) found that PBl triploids may grow betterthan PB2 triploids: however, the larval survival rateof the former may be below that of the latter.However, the available data are still inconclusive.The variation in the production of triploids may becaused by differences in experimental methods andconditions.

It is still unknown what the optimum amounts ofCB should be for blocking PBl or PB2 in the smallabalone. HaUotis diversicolor supertexta (Lischke).The small abalone is currently raised in commercialhatcheries in Taiwan (Chen & Yang 1979; Chen1984). Triploid abalone will be come an importantpart of the abalone culture industry if it has asufficiently fast growth rate. However, availabledata provide insufflcient information on the ques-tion of what the dosage of CB should be to block PBlor PB2. In the present study, karyological analyseswere used to determine ploidy in embryos, and theauthors report their initial trials in adjustingdosages of CB treatment on PBl or PB2 in thesmall abalone. It was anticipated that results of thepresent study would help to clarify the optimumconditions for inducing triploids in the smallabalone.

Materials and methods

Gamete preparation

Gametes were obtained by artificially inducedspawning of sexually mature parent abalone usingthermal variation. The abalone were separated intomale and female groups, and each group was placed

776

in a 24-L plastic c-ontainer tilled with 10 L UV-irradiated sea water. Spawning was induced bychanging the water temperature back and forthbetween 2 3 and 28 °C during a 5-h cycle. Watertemperature was increased with a heater anddecreased with water at 4 °C. Two to three cycleswere sufficient for inducing male and femalespawning. The water temperature for obtaininggametes was maintained at = 24 ± 1 °C duringspawning and fertilization.

CytochalaKin B treatment

In the cytochalasin B treatment. 1.0 mg of CB(Sigma), dissolved in 1.0 mL dimethyl sulphoxide(DMSO) as a stock solution, was added to 2L of eggsuspension (10^). and then the dosages wereadjusted to 0.25, 0.5. 1.0. and 2.0mgL"\ respec-tively. To block the release of PBl. the fertilized eggswere treated with four dosages for 10 min beginningat Jmin post-fertilization under 24 °C sea water. Toblock the release of PB2, the fertilized eggs weretreated under the same conditions as PBl. exceptthat the treatment was begun at 10 min post-fertilization. The CB was removed by collection andthe rinsing of treated eggs on a 60-|im screen. Theeggs were then returned to O.r)f. DMSO in sea waterfor JO min to remove any residual CB. In the controlgroup, the fertilized eggs were only treated withO.\% DMSO. The embryos were stocked at a densityof 5 embryos m l / ' .

Determination of the development and ploidyof embryos

Samples of embryos (n=10*) were collected 6hafter fertilization and fixed in S% formalin for thecalculation of the proportion of normal morphologyembryos. The ploidy of the resultant embryos inboth the control and treated groups was determinedby karyological analysis. Samples for karyologicalanalysis were taken at 6h post-fertilization. Em-bryos were first treated with ().or)f> colchicine for30 min. After the coichicine was removed, nineparts of a hypotonic solution ( 0 . 0 7 5 M KCI) wasadded to one part embryo suspension for JO min.The hypotonic solution was then removed, and thesamples were fixed in a solution of one part glacialacetic acid and three parts absolute methanol (v/v)for 1 h. This solution was replaced with fresh fixingsolution for an additional 1 h. Then the fixingsolution was removed and the samples were stored

© 1998 Blackwell Science Lid.. Aquacuhure Resfiinh. 29, 77S-78J

Aquaculture Research. 1998. 29, 775-783 Cytochalasin B and small abalone eggs H-S Yang et al.

in methanol at 4°C. The samples were placed in a1:1 glacial acid and absolute methanul solutionbelbre staining. The cell suspensions were droppedonto 45-50 °C slides and air dried overnight. Slideswere stained with 5% Giemsa in pH6.5 phosphatehiiffer for 3t)min and sealed with Canada balsam.These were then scanned under a compoundmicroscope for chromosome determination. In thepresent study, ploidies were classiiied as follows:(15-191 haploid; (JO-H) diploid; (45-49) triploid;(60-64) tetraploid: and (80-84) pentaplold. Allother ploidies were considered to be aneuploid.

Data analysis

Differences between the control and treatmentgroups in the proportion of normal tnorphologyand the ploidies of embryos were tested with ananalysis oi' variance (ANOVAJ.

Results

Time of release of tu'o polar bodies and firstmitosis

In the control groups. PBl was released around8 ± 1 min post-fertilization. PB2 was releasedaround 19 ± 1 min post-fertilization and iirst mitosisoccurred at 43 ± 1 min post-fertilization at 24 °C. Inthe CB-treated PBl groups. PBl was not observed inmost of the eggs at the time expected (H ± 1 min). Inthe CB-treated PB2 groups, PB2 was not observed inmost of the eggs al the time expected (19 ± 1 min).The Iirst mitosis occurred at about 50-60 min post-fertilization in the CB-treated PBl and PB2 groups.The early mitosis in the treated groups appeared tobe normal, but apparently less synchronized thanthat in the control groups.

Proportion of normal morphology ofresullanl embryos

In the control group. 98% of all eggs developed tothe gastrula stage at 6h post-fertilization, and 2%did not because of a lack of fertilization. The normalembryos were counted in both the control andtreated groups, in the control group, the normalembryo proportion was 87%. while in the PBlblocking treatment groups only 57%. 53%. 36%and 15% from eggs subjected to CB at doses 0.2 5.0.5. 1.0 and 2.0mgL"'. respectively, were nonnal.In the PB2 blocking treatment, only 5H"/o, 5b"/o,

39'fij and 6% of the embryos from eggs subjected toCB at doses 0.25, 0.5. 1.0 and 2 . 0 m g r \respectively, were normal. There were significantdifferences between the 0.2 5 and 0,5 mgL ' CB-treatment groups (/'<().05). the 1.0 and 2.0mgL"^CB-treatment groups (F<(),001). and the control,but there was no signiticani dilTerence (/'>0.05)between the PBl and PB2 CB-treatments for thesame dosage level of CB (Fig, 1), In the CB treatmentand control groups, all embryos developed toswimming trochophores at 9h post-fertilization,and the number of swimmers hatched was high inboth the treatment and control groups. Observationof the swimming ability of the trochophores showedthat the trochophores in the CB-treatment groupswere less active than those in the control group, anda higher dosage of CB was directly proportional to adecrease in the activity of swimming. The abnormaltrochophores were either deformed or constantlyswam in a circular motion on the bottom of thebeaker. In the groups with dosages of 0,25 and0.5mgL~'. and in the control group, the larvae oftrochophores developed to veligers by 20 h post-fertilization, but in the groups with dosages of 1.0and 2.0mgL"', the swimming trochophores gradu-ally died before any metamorphosis into veligers.The mitotic index of the CB-treated groups wasnoticeably lower than in control groups.

Proportion of ploidy of resultant embryos

In the control group, the karyological analysisrevealed that 41.8'K) of all cells examined had adiploid number (2n= 32) of chromosomes. Only9.0% and 7.5% could be classiiied as haploids andtripolids. and about half (41.7%) of cells wereclassiiied as aneuploids. No tetraploids or penta-ploids were found in the control group (Table 1).

Compared with the control group, all of the CB-treatment groups had increases in the proportion ofpolyploid and aneuploid cells, and decreases in theproportion of diploid cells (Tahle 1). Comparing theclassified triploids of the treatment groups, anincrease in the dosage of CB (1.0 and 2.0mgL~^)could not induce a higher proportion of triploids: incontrast, the proportion of aneuploids increased.Comparing the triploid proportions of the PBl andPB2 groups, the present authors found no signifi-cant differences (P> 0,05) for the same level dosageof CB. The majority of the cells were either diploids(10,8%), triploids (18.9%). tetraploids(5,4%) oraneuploids (51.47o) in the O.25mgL ' PBl group

© 1998 Blackwell SL-ienfc Ltd., Aquaculture Research. 29, 77S-78J 777

Cytochalasin B and small abalone eggs H-S Yang et al. Aquaculture Research, 1998. 29, 775-78 3

Control 0.25 0.5 1.0

Concentration of cytochalasin B (mg

2.0

Figure 1 The proportion of fj-h-old itormiil embryos cotinted after normal fertilization (conlroll, and blocking of the

first polar body (PBl) and second polar body I.PB2) in the fertilized eggs of the small abalune with four different dosagesof cytochalastn B ICB).

Table 1 The proportion of ploidy in the cells of 6-h-old embryos after normal fertilization (control), and blocking of the

first polar body (PBl) and the ,second polar body IPB2) in [he fertilized egg,s of the small abalone with four different

doses of cytochalasin B (CB): (In) haploid; (2nl diploid: (5[t) Iriploid; (4n) letraptoid: |5n) pentaploid; and (An)

aneuploid. Treatment yroups were compared with the control by ANOVA,

Dosage of CBtreatment (mgL~^)

Control

0.25

0.5

1.0

2.0

Polar body

PBlPB2

PBlPB2

PS1PB2

PBlPB2

Proportion ofnormal embryos

87

57*

58*

53*56-

36"

39"

15"6**

Proportion of ploidy

Sample size

67

3739

47

45

IB21

1939

1n

9.0

8.17.7

10.6*11.1'

5.64.8

5.0

20.5'

2n

41.8

10.8*15.4*

6.4*26.7

16.7*

28.6

5.0*'23.0*

3n

7.5

18.9-17.9*

12 8-17.8*

11.1

14.3'

10015.4*

4n

0

5.4

0

4.36.7

5.60

15.0

0

5n

0

5.40

12.82.2

00

5.0

0

An

41.7

51 459.0*

53.135.5

61.052,3

60 0'41.1

*'P< 0,001,

(Fig, 2). However, a high proportion of cells

examined in the CB-treated and conlrol groups

were classilied as aneuploids (Table 1). A low

proportion of haploids and pentaploids were also

recorded in the CB-treated groups. In the present

study, the Irequency of chromosDme numhers in the

778 © 1998 Blackwell Sdt Ltd., Atitiihiillurr Research. 29, 77S-7X1

Aquaculture Research, 1998, 29, 775-783 Cytochalasin B and small abalone eggs H-S Yang et al.

Fjgure2 The metaphases of (al diploid, (h) Iriploid. (c) tetraploid and (dl aneuploid cells produced by blocking the firstp()lar hddy in ihe Fertilized eggs of the small abalone wilb cytochalasin B.

control and CB-treatment groups showed a randomdistribution which indexed no relationship betweenthe lour dosages of CB and between two polar bodies(Fig. 3),

Discussion

The present study describes the results of theproportion of normal morphology and ploidy in6-h-old embryos treated with CB in fertilized eggsof the small abalone. Factors such as tempera-ture, salinity, timing and duration of treatment,and egg and sperm quality may affect the results

of polyploidy and the survival of larvae. Lu(lySfil demonstrated that different temperaturesgive dillerent yields of triploids in the Pacificoyster. Stanley etal. (1981) revealed differentproportions of polyploidy with diiTerent concen-trations of CB in the American oyster. Tabarini(1984) reported only 66% and 94'!i. triploidy ofscallops, Arejopecten irradians (Lamarck), in the().()5 and 0,1 mgL""' CB-treatments, respectively.In the above study, there was a relationshipbetween triploidy and CB concentration. However,Tabarini (19S4) did not discuss survival ratios atdiiTerent CB concentrations. There is a question

1998 Blackwell Scient-e Ltd.. Aqiiaculliirv Ri-seardi. 29, 77S-7S? 779

Cytochalasin B and small abalone eggs H-S Yaug et al.

(a)

Aquaculture Research. 1998. 29, 775-783

.r.

Control

(b)

S

4

(0-37)

a x i « s o « o n n n « » i o 2 O 3 O 4 O S O 6 O 7 O M M i o a

Chromosome number Chromosome number

(c)S

r1

(d)PB1,0.50mgr'

iI,

PBl, 1.00 mg I

10 20 30 40 SO eo ro 10 » 100 io 20 30 40 so to TO BO M 100

Chromosomt numbtr Chromotom* number

(e) (f)

£ 1

(n-20)

1

PB2.0.25mgl(n-39)

(g)

ZO 30 40 SO 60 70 80 BO 100

Chromosome number

PB2,0.50 mg I '(n-45)

10 20 30 40 H SO TO BO BO 100

Chromosome number

PB2,1.00 mg r(n-21)

J-L J -L10 20 30 40 SO 60 70 M 90 100

Chromosome number

10 20 30 40 50 60 TO 80 M 100

Chromosome number

t> 1998 Blackwell Science Ltd.. Ai{uacuhure Rcsenrch. 29. 775-78 J

Aquaculture Research. 1998. 29. 775-78? Cytochalasin B and small abalone eggs H-S Yang et al.

(i)

r

PBiiOOmgl'(n-M)

40 SO SO TOChromosama number

Figure 3 The chromosome counts in embryos after (a)tiormal fertilization (control), and (b-e) blocking of thefirst polar body (PBl) and (f-i) the second polar body(PB2) in the fertilized eggs of the small abalone with Tourdifferent dosages of cytochalasin IJ (CB),

concerning the relationship hetween tripioidysurvival and the concentration of CB. Hightriploidy and survival rates must be consideredin aquacniturc. If a high proportion of triploidywas induced by a high dose of CB, but thiscaused mass mortality of embryos, the datawould be of no value for aquaculture. Conversely.a high survival rate with only a low proportionof triploidy would also be useless. It is necessaryto find suitable parameters and to clarify opti-mum conditions for small abaionc in aquaculture.

Cytochalasin is an effective method of induct-ing polyploids in a variety of animals, Cytocha-lasin B yields a higher survival rate than heat orpressure (Downing & Allen 1987). but it is toxicto animal cells at high concentrations. Amoderate dosage of CB has been successfullyused lo inducing triploidy. In the present study,the ratio of abnormal embryos was small in thecontrol group. It is unlikely that these abnormalembryos were caused by DMSO, which is widelyused with cultured cells and emhryos. In the CB-trcatment groups, a higher ratio of abnormalitywas caused by the CB, and the results show that1,0 and 2.0mgL"' of CB are toxic to the cells ofsmall abalone, Allen etal. (1989) reported that1,0 mg L~' was used when inducing industrialtriploids of the Pacific oyster. Stanley etal. (1981)demonstrated that triploids of the Americanoyster were induced at doses of O.l-l.OmgL 'CB. In the hay scallop, triploids were induced aldoses of ().()5-(),]nigL"' CB (Tabarini 1984), Inthis study, the present authors found that someemhryos were normal at doses of 1.0-2.0mgL"'CB; however, when embryos developed intohatched swimming troehophores. all the swim-mers were abnormal. This showed that the smallabalone was more sensitive to CB than the

Pacifie and American oysters, hut less so thanthe scallop.

Duration of treatment is also an importantparameter. In this experiment, the present authorscould not explain the relationship hetween thedifference in duration and survival and ploidy.However, the interval hetween the release time ofPBl (Smin). PB2 (19 min) and (irst mitosis (4i niin)revealed that a duration of 10 min of CB-treatmentwas a suitable parameter for hlocking the release ofthe polar body. Although the 1.0 and 2,0 mgL"' CBgroups had a low proporiion of normal embryos,there may he a high normal rate if the duration timeof CB-treatment is decreased. In the present study,the percentages of normal embryos in the PBl andPB2 groups et the same dose of CB were notsignificantly different. This result conflicted withthat obtained in the Pacific oyster, in whichhlocking PBl always resulted in a higher mortalitythan hlocking PB2 (Guo el ai 1992).

The control group in the present study had adiploid chromosome frequency number of 32, inwhich there was a high ratio of aneuploids. It isunlikely that aneuploidy in the control group wascaused by DMSO, which is widely used with culturedcells and emhryos. and has no known effects onchromosome segregation. Since karyological techni-ques tend to create chromosome losses and over-estimate the occurrence of aneuploidy, a low ratemay he considered as an upper limit of aneuploidy inthe chromosome preparation. Where 4] .7% aneu-ploids were produced, the results were not oulycaused by the artifacts, but also by spontaneouschromosome segregation when the diploid chromo-some number was not equal to 2n= 32. GalTney.Scott. Koehn & Diehl (1990) suggested that hetero-zygosity deiiciency could be caused by the occur-rence of aneuploids. The formation of aneuploidy

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Cytochalasin B and small abalone eggs H-S Yang et al. Aquaculture Research. 1998. 29, 775-783

gametes in molluscs may be common, particularlyunder environmental stress (Dixon 1982). It was thepresent authors' experience with the artificialpropagation of small abalone that about half ol' thefloating larvae did not survive during the settlingstage. Haploids may result from specific failure of thesperm. It is difBcult to explain the phenomenon oftriploldy in the control group based on the presentexperimental design. Spontaneous triploids havebeen reported in amphibians, fish and molluscs andare probably caused by the fertilization of unreducedova by normal sperm (Fankhauser 194S; Thorgaard& Gall 1979; Cuoelal, 1992|.

On comparing the classified diploids of the CB-treated groups and the control group, it appearsthat the CB had acted on the cells during meiosis. tnthe present study, a high proportion of triploidyappeared in the 0.25 and 0.5 mgL"' CB groups, anda high dosage (1.0 and i.Omgir") of CH did notenhance the formation of triploidy. but increasedthe incidence of aneuploidy. It was surprising thatthe tetraploids and pentaploids were not observed inany of the PB2 groups of the CB-treatment exceptthe 0.5mgL"' group. The present authors cannotexplain this phenomenon.

The detection of triploidy after blocking PBl(18.9%) and PB2 (17.9%) in n.25mgL"' of CBagrees with results from the Pacitic oyster (15.6'X>).but the incidence of tetraploids in the present studywas only 5.4% in PBl blocked groups, as comparedwith 19.4% reported by Guo i-tal (1992). Thepresent results also differ from other studies inwhich only triploid embryos (Quillet & Panelay198(i) or tetraploid embryos (Stephens 19H9) wereobserved after blocking PBl. This variation may bea result of differences in experimental methods.conditions and animal species.

The production of a large proportion of aneuploidsby blocking PBl and PB2 was unexpected. Aneu-ploids were commonly assumed to be the result ofartifacts and CB-treatment. Guo. Hershberger,Cooper & Chew (1992) explained that the formationof aneuploidy was caused by the randomizedtripolarity and unmixed tripolarity of chromosomesegregation, an effect of CB-treatment. T^ongo,Mathews & Iledgecock (199J) also found thatchromosomes formed a tripoiar spindle duringmeiosis I after CB-treatment. As Guo et aJ. (1992)reported, chromosome tripoiar segregation inmoulds may explain the aneuploidy results owingto an unequal dyad of chromatids being lost inmeiosis. in this study, CB caused chromosomes to be

retained in the cell, resulting in a variety of ploidiesthat could be explained by a different type ofchromosome segregation. In the present study, thefrequency of chromosome number in the control andCB-treatment groups showed a random distributionthat indexed no relationship between the fourdosages of CB and the two polar bodies, demonstrat-ing that the subsequent chromosome segregation inthe cell to euploidy and aneuploidy is random. Therandomness of chromosome segregation may ex-plain this question. With regard to aneuploidy. therewas a greater opportunity for the formation ofaneuploids than other cuploids because of the CB-treatment. The results conflict with studies in thePacific oyster, where aneupoids of chromosomenumber fell into two groups in peaks betweendiploidy and triploidy. triploidy and tetrapioidy(Guo eta!. 1992). This difference in chromosomenumber distribution may a result of species differ-ences.

The proportion of normal embryos and triploidsin different dosages of CB demonstrated that resultsmay improve if dosages of CB are maintained below().2SmgL"'. and the production of a large propor-tion of aneuploids in the control and CB-treatmentssuggests that the subsequent chromosome segrega-tion is complicated.

Acknowledgments

This research was partially supported by theNational Science Council, Republic of China, andthe authors are also grateful to Dr I-Chiu Liao forsupporting this research.

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