ZHANG Zhengrui,WANG Xinglian,ZHANG Quanqi,,and Standish Allen Jr.

1) College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China,Qingdao 266003, P.R.China

2) Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, College of William and Mary,Gloucester Point,VA 23062, U. S. A.

1 Introduction

Triploid Pacific oyster (Crassostrea gigasThunberg)has become an important component of aquaculture in past decades,which is taking higher and higher proportion in total cultured oyster yield.Triploid oyster provides a year-round and high quality supply because of the reduced fecundity compared to its diploid relatives (Allen Jr.,1988).The primary method for commercial production of triploid oyster is to block the release of polar body 2 (PB2) of newly fertilized eggs with cytochalasin B (CB)(Allen Jr.et al.,1989; Allen Jr.and Bushek,1992).However,there are several disadvantages which include the limitation of the use of CB because of its potential harmfulness,the negative effect of CB treatment to the eggs and the genetic consequence caused by blocking PB2.In addition,CB treatment rarely induces 100% triploidy due to various affecting factors.These problems have been solved by crossing diploid with artificially induced tetraploid individuals (Guoet al.,1996).

In aquatic animals,some tetraploid may occur naturally(Liet al.,2011); while some others may be produced by hybridization (Xiaoet al.,2011; Liu,2010).Tetraploid can also be artificially induced by either thermal or hydrostatic pressure shock (Arai,2001; Guoet al.,2009; Yiet al.,2012).In Pacific oyster,viable tetraploid has been successfully produced by using the eggs of triploid (Guo and Allen Jr.,1994).A large amount of 100% triploid progeny are thus easily produced through reciprocal crosses between diploid and tetraploid (Guoet al.,1996).These tetraploid individuals were induced by inhibiting the release of polar body 1 (PB1) of the eggs from triploid,which were fertilized with haploid sperm (Guo and Allen Jr.,1994).Chromosome observation has proved that,except for some eutetraploid,7 types of viable aneuploid at least existed among progeny produced from the eggs of triploid (Guo and Allen Jr.,1994).

Aneuploidy in tetraploid oyster may be produced by the aneuploid gametes of their triploid parents.It has been reported that blocking meiosis by CB often causes aberrant chromosome segregation in the eggs (Longo,1972;Komaruet al.,1990; Guoet al.,1992b; Longoet al.,1993; Queet al.,1997).Some of the complex segregations were expected to give rise to aneuploid gametes(Longo,1972,Komaruet al.,1990; Guoet al.,1992a;Guoet al.,1992b; Longoet al.,1993; Guo and Allen Jr.,1994; Queet al.,1997).Aneuploidy in tetraploid may also originate from aneuploid gametes produced by normal segregations in aneuploid or heteroploid mosaic individuals of their parents.Some triploid individuals have been reported to lose approximately one set of chromosomes in part of their cells and revert to heteroploid mosaics containing cells with different numbers of chromosomes (Allen Jr.et al.,1996; Zhanget al.,2010a).Cytogenetic analysis has revealed that some ‘triploid’ individuals are actually aneuploid with 28,29 or 31 chromosomes instead of 30 of triploidy (Wanget al.,1999;Zhanget al.,2010a); while all the mosaics gave a high percentage of hypotriploid cells (Zhanget al.,2010a).Meiotic chromosome observation revealed that a considerable proportion of aneuploid cells could enter normal meiotic process and aneuploid gametes could be produced if these cells go through gametogenesis (Zhanget al.,2010b).Aneuploid gametes may also be produced by aneuploid cells in some diploid individuals.On the other hand,polyploids are unstable and some are reversible (de Wet,1971).Triploid oyster can revert to heteroploid mosaics in field through chromosome elimination (Zhanget al.,2010a).Some tetraploid individuals have been found as heteroploid mosaics after two years of culture (Allen Jr.et al.,1996,Guoet al.,2009).This implied possible reversion exists in tetraploid oyster as does in triploid oyster.

The adult tetraploid individuals were all isolated by flow cytometry before they were used for triploid seed production.Yet,it is difficult to detect small differences in DNA content with flow cytometry.In fact,Guo and Allen Jr.(1994) found a certain proportion of aneuploid individuals by chromosome observation.Since aneuploid individuals and individuals with aneuploid cells are viable in Pacific oyster (Thiriot-Quievreuxet al.,1988; Thiriot-Quievreuxet al.,1992; Guo and Allen Jr.,1994; Zouroset al.,1996; Wanget al.,1999,Zhanget al.,2010a),it is possible that the adult tetraploids isolated by flow cytometry might include aneuploid individuals and/or heteroploid mosaics that have a few chromosome differences.

Guoet al.(1996) reported that crosses within tetraploids gave lower survival than crosses within diploid and between diploids and tetraploids.This result was attributed to genetic defect of inbreeding of tetraploids used in the crosses and the possible aneuploidy of the gametes derived from the meiotic segregation of tetraploids (Guo and Allen Jr.,1997).It is also possible that the aneuploid gametes may be produced by aneuploids or heteroploid mosaics reverted from eutetraploids as was reported in aneuploids or heteroploid mosaics which reverted from eutriploids (Zhanget al.,2010b).

Up to date,it has been ascertained that not all the adult survivors of ‘tetraploid oyster’ are eutetraploids with exactly 4 sets of chromosomes in all cells.Some individuals may be aneuploids; whereas others may be mosaics either with two different kinds of cells or with various kinds of cells possessing different numbers of chromosomes (Allen Jr.et al.,1996).However,the reasons for the occurrences of these kind individuals remain unknown.Therefore,the objective of the present study was to investigate the ploidy status of the adult ‘tetraploid oyster’ through detailed cytogenetic observation and with flow cytometry to insight into the mechanism of chromosome loss–the reversion in tetraploid oyster.Cytogenetic evidence for the origin of a high proportion of aneuploid individuals and heteroploid mosaics was presented and discussed.

2 Materials and Methods

Two years old tetraploid oyster was produced by inhibiting the first polar body (PB 1) release of the eggs of triploid females fertilized with spermatozoa of normal diploid males CB (Guo and Allen Jr.,1994).Oyster was gaped by overnight immersion in 7‰ MgSO4.A small piece of gill biopsy was dissected from each individual for flow cytometric screening (Ploidy Analyzer PA-I,Partec) and another piece for chromosome preparation.Flow cytometry was conducted in accordance with Allen Jr.(1983).Chromosome samples were prepared as was described early (Zhanget al.,2010a).

Two or three gill filaments were taken to a microcentrifuge tube containing several drops of fresh fixative,chopped with sharp forceps and pipetted gently with Pasture pipette to make cell suspension.Then the cell suspension was dropped onto a slide glass cleaned with 70%ethanol and air-dried.The slide was then stained with 5%Giemsa in pH 6.5 phosphate buffer for 15min,observed under microscope and photographed.

The mitotic chromosome numbers of the metaphase spreads showing no obvious chromosome loss and/or overlaps were counted to clarify the chromosome status of each individual.At least 10 cells from each individual were recorded.

3 Results

Using a wild diploid as control (Fig.1a),20 individuals were isolated by flow cytometric screening from the batch (Table 1).Among them,17 showed a single tetraploid peak (Fig.1b),1 individual (#18) showed a continuous distribution of cells from triploid to tetraploid but peaked at tetraploid range (Fig.1c),and the remaining 2(#19,20) were tetraploid/triploid mosaics (Fig.1d).

Unambiguous metaphase spreads were obtained from gill biopsy of all the 20 oyster individuals.Chromosome counts showed that 9 of the 20 (45%) individuals were eutetraploids that had a mode of cells with 40 chromosomes (Fig.2a); 2 (10%) were aneuploid,one (#10) showed a majority of cells with 39 chromosomes,and the other one (#11) with 38 chromosomes (Table 1,Fig.2b,c).The remaining 9 (45%) individuals were heteroploid mosaics.For individual #12,50% of its cells contained 40 chromosomes and the other 50% of cells contained 39 chromosomes (Table 1).Chromosome counting result of indi-vidual #18 coincided with flow-cytometric analysis.Of the 67 cells observed in individual #18,27 cells showed 40 chromosomes that constituted the largest cell population,but 25 aneuploid cells in total were observed exhibiting various numbers of chromosomes ranging continuously between triploid and tetraploid.The result of chromosome counting for the 2 mosaic in dividuals coincided with their flow cytometry analysis.However,the other 5 individuals (#13–17) were also identified as heteroploid mosaics mainly containing tetraploid cells,while various proportions of triploid and aneuploid cells were also found (Table 1,Fig.2e).Three individuals (#14,15 and 17) even had some diploid cells (Table 1,Fig.2f).The percentage of triploid and diploid cells varied significantly among different individuals.Most of the individuals had more tetraploid cells than triploid and diploid cells,while individual 20 had more triploid cells (Table 1,Fig.1d).Karyological analysis for the randomly selected several cells with 40 chromosomes revealed normal 4 sets of chromosome constitutions in these cells.

Fig.1 Flow-cytometric histogram of diploid control (a),tetraploid individual 1 (b),heteroploid mosaic 18 (c) and tetraploid/triploid mosaic 20 (d) of Pacific oyster.

In individual 1,all the 10 cells observed had 40 chromosomes.In contrast,all the other 19 individuals contained various numbers of aneuploid cells ranging from triploid to tetraploid.Most of these aneuploid cells were hypotetraploid cells exhibiting 35–39 chromosomes (Table 1).Although a majority of cells at triploid range with 29–34 chromosomes were eutriploid cells in total,hypertriploid cells were also observed in most of the heteroploid mosaics (Table 1,Fig.2d).In individuals 17,18 and 19,eutetraploid cells only consisted 22.7% (5 in 22),40.3% (27 in 67) and 32.3% (10 in 31) of total cells,while aneuploid cells consisted 77.2%,37.3% and 38.7%of total cells,respectively.

In 10 (50%) individuals,certain number of metaphase spreads showed 32–37 well-scattered and some coagulated chromosomes (Table 1,Fig.3a–c).These chromosomes associated together to make a chromosome clump.In a few cases two chromosome clumps were observed in one cell.The chromosome clumps exhibited the same characteristics as those observed in mosaics of triploidC.gigasandC.ariakensis(Zhanget al.,2010a).In some cells it was able to detect the number of associated chromosomes in a clump,generally 3–8 chromosomes.But in most cases it was difficult to detect the exact number of chromosomes.One cell in individual #16 showed obvious asynchronous chromosome condensation.Only about 30 chromosomes condensed normally,while other chromosomes were improperly condensed (Fig.3d).

Notably,among the 10 individuals that showed chromosome clumping,only two were eutetraploid individuals whereas all the other 8 individuals were heteroploid mosaics.Individual showing higher proportion of total aneuploid cells between triploid and tetraploid tended to have more cells with chromosome clump(s).The correlation between the two categories of cells among the 8 heteroploid mosaics was significant (P<0.05) (Fig.4).

Fig.2 Chromosome constitutions in gill cells isolated from tetraploids and heteroploid mosaics.a),A cell from a eutetraploid individual showing 40 chromosomes.b),A cell from an aneuploid individual showing 39 chromosomes.c),A cell from an aneuploid individual showing 38 chromosomes.d),A hypertriploid cell with 32 chromosomes.e),An eutriploid cell from individual 15 with 30 chromosomes.f),An eudiploid cell from individual 15 with 20 chromosomes.

Table 1 Frequency of cells with various numbers of chromosomes in gill tissue of tetraploid C.gigas isolated by FCM from the batch produced by blocking PB1 of the eggs of triploids fertilized with haploid sperms

Fig.3 Cells with various numbers of well-spread chromosomes and chromosome clump (arrows) (a–c),and one cell showing about 30 normally condensed chromosomes and some asynchronously condensed chromosomes (arrow heads)(d).

Fig.4 Positive correlation between the percentage of cells with chromosome clumping and that of total aneuploid cells.The squares and solid line shows the analysis with data from only heteroploid mosaics (P<0.02).The triangles and dash line shows the analysis with data from all individuals (P<0.05).

4 Discussion

Guo and Allen Jr.(1994) have found several types of aneuploids in the ‘tetraploid’ oyster.About 17% of the tetraploids identified by flow cytometry in their population were actually hypo- and hyper-tetraploids.In this study,we observed a high proportion of aneuploids and heteroploid mosaics among the adult ‘tetraploid’ Pacific oyster induced from eggs of triploids.Of the 20 individuals that were isolated as tetraploids by flow cytometry,9(45%) could be categorized as eutetraploids; 2 (10%)individuals were apparently aneuploids and the remaining 9 (45%) were heteroploid mosaics.Hyper-tetraploids were not found in our observation.But the total percentage of aneuploids and mosaics were much higher than the reported by Guo and Allen Jr.(1994).Viable heteroploid mosaics or aneuploids are very rare in vertebrate animals such as fish (Davissonet al.,1972; Zhang and Arai,1999;Zhang and Arai,2003).In oyster,however,somatic aneuploidy and viable aneuploids have often been reported(Thiriot-Quievreuxet al.,1992; Guo and Allen Jr.,1994;Zouroset al.,1996; Wanget al.,1999; Gonget al.,2004;Allen Jr.et al.,1996; Guoet al.,2009; Zhanget al.,2010a; this study).Aneuploidy in tetraploids may originate from the aneuploid gametes produced by aneuploid or heteroploid mosaic individuals in triploids (Guo and Allen Jr.,1994).Observations of chromosome behavior during meiosis in triploids and mosaics revealed that considerable proportion of aneuploid cells could enter normal meiotic process,implying the production of aneuploid gametes through gametogenesis of these cells (Zhanget al.,2010b).The high incidence of heteroploid mosaics observed in this study,however,cannot be explained by the aneuploid gametes.This result strongly suggested that reversion also exists in tetraploid Pacific oyster,and the heteroploid mosaics are originated from tetraploids as having been reported before (Allen Jr.et al.,1996; Guoet al.,2009; Zhanget al.,2010a).

In our early study,we have found high percentages of hypotriploid cells and cells with chromosome clumping in the mosaic individuals of bothC.gigasandC.ariakensis.The significant positive correlation between these two categories of cells lead us to conclude that chromosome clumping is the mechanism for the chromosome loss in triploids of both species (Zhanget al.,2010a).Similar to the results observed in mosaics of triploidC.gigasandC.ariakensis,a considerable number of aneuploid cells were observed in most of the tetraploid individuals sampled.Chromosome clumping was detected in 10 individuals,of them 8 were heteroploid mosaics.The percentage of aneuploid cells with chromosome clump correlated significantly with that of aneuploid cells with chromosomes varying between triploid and tetraploid (P<0.02).These results provided further evidence that chromosome clumping is the mechanism for the chromosome elimination in polyploid oysters.One cell showed obviously asynchronous chromosome condensation.It is difficult to determine whether the asynchronous condensation correlates with chromosome clumping or chromosome elimination in this species,because of the low frequency of this kind of cells.However,we believe that this abnormal chromosome behavior would subsequently affect the later mitotic chromosome segregation and result in daughter cells with unusual chromosome constitutions.Chromosome elimination through improper chromosome condensation has been reported in some insect hybrids (Ryan and Saul,1968; Johnanneset al.,1990).

Several individuals showed not only tetraploid and triploid cells,but also diploid cells.This suggested that cells in tetraploids may not stop their chromosome elimination process when they revert to triploid state.Some of the reverted triploid cells may continue to eliminate their chromosomes until they reach diploidy,which is the most stable ploidy state for bisexual animals.This fact in tetraploid oyster proved again that chromosome elimination is a new mode of rediploidization in polyploid oyster which differs from other modes reported in some ancestral polyploid animals (Allendorf and Thorgaard,1984;Ferris,1984).

Polyploids are not stable in plants (Snoad,1955; Lewiset al.,1971; Tan and Dunn,1977) and yeast (Mayer and Aguilera,1990).Some polyploids can revert to diploid status to return to the natural stable genome balance(Randolph and Fisher,1939; Kimber and Riley,1963; de Wet and Harlan,1970; de Wet,1971; Zhang and van der Meer,1988).Polyploids are very rare and especially unstable in higher animals (Darlington,1953).Even polyploid cells in mammals are genetically unstable and often result in aneuploidization and aggressive cell divisions or tumorigenesis (Ornitzet al.,1987; Shackneyet al.,1989;Levineet al.,1991; Andreassenet al.,1996).The previously reported reversion of triploidC.gigasandC.ariakensis(Allen Jr.et al.,1996; Zhanget al.,2010a) as well as the reversion of tetraploidC.gigasobserved in the present study suggest that reversion is a common characteristic in polyploid oyster.

As demonstrated in Table 1,individual 12 was a heteroploid mosaic with half of its cells showing 40 chro-mosomes while the other half showing 39 chromosomes.Absolutely no other hypotetraploid cells and chromosome clumping were observed.It was different from the other mosaic individuals that showed some chromosome clumping and wide range of aneuploid cells with various numbers of chromosomes.Although the present results could not rule out the possibility that this mosaic is derived from reversion of a eutetraploid,it seemed less likely for an individual to eliminate strictly one chromosome from 50% of its cells during somatogenesis.Therefore,we considered this individual to be ‘intrinsic’ mosaic that was either derived from abnormal fertilization or developed during early embryogenesis.

Two hypotetraploid aneuploids were detected with 39 and 38 chromosomes respectively.Hypertetraploid aneuploids were not identified.This may be attributed to the small sampling size.The origin of the two aneuploid individuals is also difficult to determine.Similar to the reasons discussed above,these aneuploids may have developed from aneuploid gametes produced by triploid and/or diploid heteroploid mosaic parents that were aneuploids or contained large number of aneuploid cells (Zhanget al.,2010a).They may have been produced by abnormal chromosome segregation of the eggs caused by CB treatment during tetraploid induction (Longo,1972; Komaruet al.,1990; Guoet al.,1992b; Guo and Allen Jr.,1994;Queet al.,1997).Whatever the origination mechanisms of aneuploids and heteroploid mosaics,the variability in chromosome constitution of tetraploids existed and the total proportion of these individuals in tetraploids were very high.

The variation of chromosome numbers in parents will yield gametes with varying numbers of chromosomes,and the variation in chromosome numbers within and among progeny groups can be expected.Guoet al.(1996)reported lower survival in crosses within tetraploids,and attributed it to inbreeding or the possible aneuploid gametes produced by meiotic segregation of tetraploids.The existence of high proportions of aneuploids and heteroploid mosaics identified in the present study provided us new evidence that aneuploid gametes may also be produced by these aneuploid or mosaic parents through twisted or normal segregation.This also suggested different genetic basis of reversion in different triploid groups.Since aneuploidy often causes greater genome instability (Sandberg,1977; Babinovitchet al.,1989;Mayer and Aguilera,1990; Giaretti,1994),it is virtually reasonable to link the difference in the rate of reversion with the variation in chromosome numbers among triploid groups derived from different tetraploid parents.Therefore,the relationship between reversion in triploids and the aneuploidy and/or mosaicism of their ‘tetraploid’parents is of great interest deserving further detailed study.

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