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International Council C.M. 2002/U:11for the Exploration of the Sea Mariculture Committee

Trends in genetics of bivalve mollusks: A review

S. Stiles and J. ChoromanskiAbstract

Recent advances in genome mapping and the development of linkage maps, as well as theproduction of polyploids and aneuploids, in bivalve mollusks have led to a resurgence of interestin chromosome organization and manipulation. Knowledge of normal meiosis and baseline dataon mitoses in these invertebrates can assist in such undertakings and serve to elucidate genomefeatures and processes. Baseline data will be presented on meiosis and genetic manipulationsuch as polyploidy, aneuploidy, and tetraploid cloning in the eastern oyster, Crassostreavirginica. For example, approximately 12% of eggs from mass-spawned populations of C.virginica oysters were heteroploid. Haploids were 6%, polyploids 1.5%, hypodiploids 1.5%,hyperdiploids 1.5%, and mosaics 1.5%. Thus, aneuploids averaged 3% in these populations ofoysters, which can serve as a reference frequency for current studies on aneuploidy in easternoysters. Previous chromosome engineering efforts for the induction of cloning or polyploidy in15 experiments with eastern oysters revealed that the ploidy level of early embryos developedfrom eggs treated with cytochalasin B, high pressure and/or exposed to irradiated sperm ingeneral ranged from haploidy through pentaploidy. Outcomes depended on the female,experimental conditions, synchronous development and whether or not the sperm weregenetically inactivated with irradiation. Triploidy occurred as high as 66%, but generally rangedfrom 3% to 38%. Some embryos were chromosomal mosaics or aneuploids. Implications forgenetic manipulation, including transgenesis, in other bivalves such as bay scallops will be discussed. Results should be considered with regard to efforts for rehabilitating or restockingbivalve populations.

Key Words: Chromosomes, polyploidy, aneuploidy, cloning, bivalve mollusks, genomemapping, linkage

S. Stiles and J. Choromanski, National Oceanic and Atmospheric Administration(NOAA)/National Marine Fisheries Service (NMFS), Milford Laboratory, Milford, Connecticut,USA (Tel. 203-882-6524; FAX 203-882-6517; email: sstiles@clam.mi.nmfs.gov,jchoromanski@clam.mi.nmfs.gov

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INTRODUCTION

New developments in genomics are a key component of modern genetics with some understanding of basic concepts such as genome evolution and gene interactions. They alsoprovide a basis for the application of modern genetics to biological, agricultural and aquaculturesciences, including biotechnological advances. For example, relationships are being exploredfor DNA replication, Mendelian ratios and Hardy-Weinberg equilibrium. Genomic sequencedatabases are also being explored, providing new insights into how genes function as well ascharacterization of transposable elements. In addition, an understanding of dimensions betweenDNA and chromosomes is becoming necessary. However, such relationships between physicaland genetic distances of genes and their impact on genetic events, gene expression, andevolution have been little elucidated.

Trends in genetics of bivalve mollusks encompass investigations of genome mapping andlinkage, the development of inbred lines to subsequently cross for heterosis, the development ofhybrids and the induction of polyploidy and aneuploidy. Application of molecular (DNA) andcytological (banding) tools are valuable for population genetics investigations of stockidentification and related studies on marker-assisted selection and quantitative trait loci forbreeding. Ultimate goals are increased harvests of natural populations and increased productionin commercial hatcheries.

Aquaculture or controlled culture of bivalve mollusks has provided opportunities forselection and breeding to improve stocks. Approaches have included inbreeding, mass selection,genome manipulation and population analyses for genetic diversity. Overall, some progress hasbeen made through selective breeding for commercially important traits such as growth,survival, and disease resistance. Recent advances in genetics of some commercially valuablebivalve mollusks include induction of triploidy, tetraploidy and aneuploidy, and development ofdisease-resistant lines in oysters, development of notata clams as genetically-marked stocks, anddevelopment of genetically-marked and transgenic lines of scallops. Since gametes of bivalvesgenerally are spawned or released at metaphase I of meiosis, opportunities also exist and areafforded for use of genetic manipulation or biotechnology, not easily done in finfish or othervertebrates. In addition, millions of gametes can be manipulated for polyploid, cloning andtransgenic induction.

The purpose of this present review, primarily of chromosomal genetics using the easternoyster (Crassostrea virginica) as a model, was to evaluate status and trends including genomemapping and, particularly, cytological responses of a bivalve to some common means forinducing gynogenesis, androgenesis and polyploidy. The value of genetic manipulations such asgynogenesis in practical breeding and in basic research has been pointed out by Purdom (1983),Kirpichnikov (1981), Stanley (1974) and several researchers as listed in the bibliography. Inaddition, production of successful gynogenetic progeny offers some special opportunities forstudying the quantitative inheritance of economic traits.

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Chromosome Genetics

One of the bases to a better understanding of these approaches involves chromosomalvariation and polymorphisms or genomic processes in evolution or selection. Concurrent withadvances in molecular genetics has come increased awareness and understanding of the role ofchromosomes as a significant part of genomic research in any organism. Additionally, theadvent of molecular marker techniques has greatly facilitated the construction of linkage maps. For example, linkage maps have been constructed using morphological traits, isozymes,restriction fragment length polymorphisms, (RFLPs), random amplified polymorphic DNAs(RAPDs), and microsatellite DNA. An important application of linkage maps is that these serveas a starting point for the identification of quantitative trait loci. As more genes are identified,mapped and linked to specific traits, genetic and chromosomal assays will take on newimportance. Early development of maps was difficult due to the paucity of marker loci and togenotype environmental interactions which can modify the expression of qualitative traits. Inaddition, map utility has often been constrained by the lack of linkages to economicallyimportant traits.

Genome Mapping

Genome maps are created from markers - DNA sequences in or near genes whoselocations are known - and compared with the occurrence of favorable traits. If the markers andtraits appear together more often than would occur by chance, the locations of the genes for thedesirable trait are likely to be near the markers. Multiple genes usually govern a single trait ofeconomic importance. The locations of these genes are called quantitative trait loci or QTLs. Once QTLs are identified, DNA tests are conducted on breeding lines to find out whether theyhave the desired QTLs. If so, marker - assisted selection (MAS) enables the researchers to putthese traits into new breeding programs much sooner than if they used trial and error breeding toidentify organisms with good genes. Comprehensive genome analyses should entail analyzinggenome rearrangements in the context of cytogenetics, molecular genetics, population biologyand breeding projects.

Advances in the characterization of bivalve chromosomes can contribute to genomemapping. Early studies involved karyotyping and chromosome banding with some degree ofsuccess. More recent analyses include computer-assisted karyotyping (Zhang et al, 1999).Early attempts to map the genetic basis of heterosis in bivalves employing quantitative trait lociwere met by problems from distortions of Mendelian segregation ratios, as reviewed by Launceyand Hedgecock (2001). Based on microsatellite loci, these investigators reported distortedsegregation ratios in Pacific oyster families and estimated the genetic load. It was hypothesizedthat selection against recessive deleterious mutations at closely linked genes was responsible fornon-Mendelian inheritance of markers, beginning in the juvenile stage. They concluded thatsuch distortions resulted from homozygote disadvantage rather than heterozygote advantage. These could be caused by a high mutation rate or by association of a large number of fitnessgenes with a few markers in a small genome. The haploid number of chromosomes in allCrassostrea and Ostrea species studied thus far is 10.

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Genomes can evolve by acquiring new sequences and by rearranging existing sequences. Another source of variation is transposable elements or transposons, which are sequences in thegenome that are mobile. Recombination also is a key event in the evolution of the genome. Recombinant chromosomes contain different combinations of alleles, providing the raw materialfor selection. One important mechanism for the genome to change its content of genes ratherthan a combination of alleles is crossing-over, or when a recombination event occurs betweentwo sites that are not homologous. Crossover events in meiosis can be observed in bivalves asdemonstrated in the prometaphase I or diakinesis group and metaphase I bivalents in Figures 1aand 1b.

Chromosome Manipulation

The development of triploid and tetraploid organisms has been a significant componentof agricultural potential to enhance aquacultured species. While progress has been made in thearea of chromosome manipulation, consistent results have not always been achieved. Basicapproaches in bivalves include induction of triploidy, tetraploidy, gynogenesis, androgenesis andcloning.

Chromosome manipulation or engineering could play a significant role in the aquacultureof bivalves. Products could range from gynogens or clones of organisms with outstanding traitsto polyploids which also could be commercially valuable. Gynogens could be produced inbivalve mollusks by stimulation of the egg to proceed through meiosis without fusion with thesperm or male contribution. Variation on the process could involve destruction of the oocytenucleus with development of the male nucleus in the oocyte, a process known as androgenesis. Parthogenesis is the general term for female or male development without syngamy or thecontribution of either sex. Doubling of the haploid chromosomes then would result in a diploidgynogen, or the equivalent of a clone of the female or male exempt crossing over.

Unlike eggs of finfish, shellfish eggs divide holoblastically, therefore, their immediatepost-fertilization chromosome stages can be studied with considerable ease and very reliably(Longwell et al., 1967; Longwell and Stiles, 1968a; Stiles and Longwell, 1973). Because thespawned, unfertilized shellfish oocyte is blocked at Metaphase I of meiosis, not Telophase II asin finfish, it affords opportunities to study and to manipulate even earlier meiotic stages thanpossible in finfish.

Most oysters discussed in the following studies were from wild Crassostrea virginicastock local to the vicinity of the harbor of New Haven, Connecticut. Eggs and the sperm used tofertilize them came from thermal-induced spawnings of adult male and female oystersconditioned in the laboratory (Loosanoff and Davis, 1963).

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Regular Meiosis and Fertilization in Spawned Eggs of the Eastern Oyster

Normal, unfertilized, spawned eggs of C. virginica are either at prometaphase ormetaphase I of meiosis. Occasionally a spawned egg is at diakinesis (Fig. 1a) or late diplotene. Few unfertilized eggs proceed beyond metaphase I. (Fig.1b).

Karyotype studies on early cleavage chromosomes of this oyster (Longwell et al., 1967)have shown them to range from 2 to 3.5 microns long at colchicine metaphase. Oysterchromosomes consist of 10 homomorphic isopyknotic pairs. The genes occur linked in 10different groups. Meiotic chromosomes have some chiasmata, an indication of crossing over ofgenes. Therefore, linkage of the oyster’s genes is not complete. Meiosis of the egg generally isof the usual type with genetic segregation. Similarly, meiotic divisions in the male gonad appearto be normal.

Irregular Meiosis and Fertilization in Spawned Eggs of the Eastern Oyster

Various deviations from the usual sequence of events of meiosis and fertilization werenot infrequent in the material used in these studies. Aneuploid, haploid, triploid, andchromosomally mosaic embryos are the result of such disturbances. They no doubt lead to poorembryo and abnormal larval development, and death in a significant number of cases.

Polyspermy can occur in C. virginica and more than one sperm nucleus can be resolvedinto chromosomes in a single egg. These extra nuclear groups can either remain in situ in thecytoplasm or several of them may fuse with the female group with the resultant formation of amultipolar spindle.

In 17 different mass spawnings of a total of 835 wild Long Island Sound oysters, acombined total of 6% of 1724 early cleavage eggs examined chromosomally were spontaneoushaploids. Spontaneous polyploidy occurred at a much lower frequency, about 1.5%. Aneuploideggs (those with a few extra or missing chromosomes) were about 3% of all examinedcytologically, and mosaics, about 1.5%.

Percent Numerical Chromosomal Abnormalities in Wild Oyster Populations (C. virginica)

Percent

Haploidy 6

Polyploidy 1.5

Aneuploidy 3

Mosaicism 1.5

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Experimental Manipulation with X-Irradiation

Parthenogenesis

For some time it has been known that sperm can be genetically inactivated throughdestruction of its chromosomes by irradiation, yet remain physiologically viable and activeenough to stimulate development of the egg which it penetrates. This is a special case ofparthenogenesis termed gynogenesis. Induced parthenogenesis, if practical or possible in theoyster, would circumvent the necessity of breeding several oyster generations to obtain highlyinbred individuals and lines. Accordingly, the effectiveness of X-irradiated sperm in inducingparthenogenesis in the oyster was tested.

For the induction of parthenogenesis, spawned sperm in seawater was irradiated using aSiemens Stabilpan X-ray especially calibrated for the high doses necessary for the invertebratesperm. Eleven doses, ranging from 10,000 R to 225,000 R, were delivered to the oyster sperm ininitial efforts to determine which dose might best induce parthenogenesis. Cytogenetic criteriafor determining parthenogens were based primarily on progression of meiotic stages of the egg,i.e., anaphase I, metaphase II, anaphase II or pre-fusion, without the normal progression ofstages of male development. Spawned, unfertilized eggs of C. virginica usually remain atMetaphase I or the stage just preceding the ones mentioned above. In full-sib crosses of theeastern oyster, the spontaneous incidence of parthenogenesis appeared to be increased greatly(Longwell and Stiles, 1973).

True induced haploid parthenogenesis, as measured cytologically, ranged from 4% with15,000 and 225,000 R to the spawned sperm, to 15% with 150,000 R. Spontaneousparthenogenesis averaged only 1% in the control in accordance with earlier measurements(Longwell and Stiles, 1968; Stiles and Longwell, 1973). Development at 10,000 R represented66% true fertilization, with the X-irradiated sperm contributing severally damaged chromosomesto the zygote. Similarly, at 15,000 R, the sperm participated in fertilization, producing cleavingembryos in 19% of the eggs. Ninety-six percent of the eggs cleaved. At 15,000 R, as many as13.6% of the cultured eggs developed to the straight-hinge larval stage and in the control, 24%. Development to straight-hinge at 20,000 R was 1.7%, 3.9% at 25,000: at 100,000 R, 2.4%; at175,000 R and 225,000 R, 2.9% and 2.8%. It is uncertain if any of the straight-hinge larvae weredeveloped from parthenogenic eggs. Induction of successful gynogenesis in oysters withirradiated sperm does not appear to display a clear Hertwig effect, and may not be so readilyobtainable as in fish. It needs further investigation to appraise its practicality. Spontaneousparthenogenesis in the oyster appears to increase with inbreeding (Longwell and Stiles, 1973b)Possibly, inbred lines could be used to some advantage in this regard.

More trials are needed to pinpoint better a dose of irradiation necessary for the geneticinactivation of oyster sperm intended for parthenogenetic stimulation of oyster eggs. It seemsthat the lower doses of 10,000 R and 15,000 R could be eliminated from further testing sincethese doses allowed true fertilization to occur. There was a significant decrease in developmentof eggs to the straight-hinge larval stage at 20,000 R. This could indicate a point of maximum

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development problems resulting from contributions of damaged male chromosomes to the eggsin true fertilization. Doses from about 30,000 R or even higher than 225,000 R should be furthertested.

Complicating the determination here is a sperm dilution factor, which can vary fromexperiment to experiment. The small size of the oyster sperm and the relatively small number ofits chromosomes may be factors contributing to its overall sperm sensitivity to irradiation. Sperm from the surf clam at dilutions of one to 2,000 could not tolerate exposure above 31,500R, compared to effective doses of irradiation as high as 264,000 R delivered to dry orconcentrated sperm (Rugh, 1953). Irradiated sperm in an aqueous medium may produce moreobscure effects because of some interaction between products of irradiation and the mediumalone.

Manipulating Division of Meiosis I to Clone Maternal Genomes of Shellfish

If shellfish eggs naturally blocked at meiotic metaphase I until fertilization are inhibitedfrom entering anaphase or the two products of this division fused, and gynogenesis induced withgenetically inactivated sperm, the maternal genotype can be conserved in any resulting,chromosomally normal embryos. The tetraploids among these have all the maternal genes, anddiploid ones, partial copies of maternal genes. Experimental outcomes of disruptions of meiosis I in the eastern oyster, Crassostrea virginica Gmelin, are presented, and prospects fordeveloping a reliable technology to multiply the maternal genome of selected shellfish areevaluated. In addition, evidence is presented for the series of chromosome events that mustoccur in the production of triploids.

More than 40 experiments were conducted at a constant temperature of 25°C forchromosome manipulation in oysters. Some results came from preliminary trials to determinethe optimal dose of UV-irradiation to the sperm and optimal treatment of pressure on eggs forinduction of gynogenesis and polyploidy, respectively. Results from approximately 15representative experiments on the various treatments for induction of the more heterozygous“clones” are discussed.

Cytological Assessment of Experimental Manipulation with Ultraviolet Radiation, CytochalasinB and High Pressure

Genetic manipulations of chromosomes in bivalve shellfish generally have concernedinduction of either gynogenesis for developing genetically homozygous lines or of polyploidyfor obtaining sterile triploid strains. However, manipulation of meiosis I in bivalves, in contrastto manipulation of meiosis II in finfish (Streisinger et al., 1981), could also result inheterozygous genotypic copies of selected superior shellfish. This can be accomplished byinduction of gynogenesis and suppression of meiotic divisions. The focus in this case is notincreased homozygosity as selfing connotes, but the production of copies of a superior maternalgenotype in the heterozygous state when maternal and paternal homologues are retained as theyare found in the sole female parent. This outcome can likely occur in all organisms such as anoyster in which eggs are available in metaphase I of meiosis and therefore for suppression of

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either or both maturation divisions. Chromosomal evidence for these possibilities would be thepresence of pentaploids, tetraploids, triploids, and diploids with 0, 1 or 2 polar bodies.

Cytological observations can demonstrate the existence of these karyotypes and modelscan be derived from the direct microscopic observations. Results could also explain differentialheterozygosity of triploids depending upon whether Meiosis I or II is suppressed (Stanley andAllen, 1984). For example, if heterozygous triploids can be produced by cytochalasin B or highpressure treatment of normally fertilized oocytes some pentaploid embryos are also expected tooccur based on models of behavior at meiosis I. Moreover, similar treatment of oocytesfertilized with genetically inactivated sperm should result in some non-inbred diploid ortetraploid replicas of the female parent with the exception of cross-over segments.

Gynogenesis and parthenogenesis have already been investigated in bivalves with somesuccess when heat, X-irradiation, UV-irradiation or ionic and chemical solutions were used(Morris, 1917; Allen, 1953; Rugh, 1953; Stiles, 1978; Stiles et al., 1983). In addition, inductionof polyploidy in bivalves seems to offer some probability of success (Longwell, 1968, 1985,1986; Longo, 1972; Allen and Stanley, 1981; Stanley et al., 1981).

This particular study concerns a series of experiments conducted on the eastern oyster,Crassostrea virginica, in which techniques for gynogenesis and polyploidization were combinedand effects assessed cytologically. The prime purpose was to identify and quantify those eventswhich could lead to production of near identical maternal genomes or clones. Another purposewas to elucidate cytological events which must occur in procedures used with varying success toproduce triploid shellfish.

Treatments for Inducing Gynogenesis and Polyploidy

Ultraviolet radiation of sperm for the purpose of its genetic inactivation was conductedwith a short-wave UV lamp (254 nanometers) of 8 watts contained in a quartz envelope. Spermsuspensions of 50 ml were held for treatment in glass Petri dishes placed 15 cm from the lightsource. The concentration of sperm as measured by a hemocytometer was 4.2 X 106 per ml inseawater. Sperm exposed for 3.5 minutes had the greatest efficiency in inducing gynogenesis toeggs. Dose of UV-radiation delivered to sperm was 100 ergs/mm2/sec.

Initial density of eggs at the time of fertilization was 1500/ml in a 1 liter volume ofseawater. This density was reduced to 750 eggs/ml when the first sample was taken for cytologyand an aliquot was removed for culture within the first hour after fertilization. By 8 hours post-fertilization the concentration of eggs was 375/ml. Density for culture to 24-48 hours wasmaintained at a usual 30 eggs/ml.

Eggs subjected to pressure treatments were held in small compartmentalized steelcontainers placed inside an alloy steel pressure vessel within 1-5 minutes after the addition of thesperm. Eggs were subjected to high pressure treatments ranging form 6,000 - 14,000 p.s.i. for 5-10 minutes with the best results obtained at 12,000 p.s.i. for 5 minutes. Other fertilized eggswere exposed for approximately 15 minutes to cytochalasin B at a concentration of 0.5 mg/l in

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0.005% dimethyl sulfoxide (DMSO) solution. Ploidy levels of eggs, embryos, and larvaesampled at various times during development were determined by their direct cytologicalexamination.

Induced Gynogenesis

Irradiation of oyster sperm for 2.0, 2.5, 3.0 and 3.5 minutes had no effect on its ability tofertilize oyster eggs, and the treatment did not cause polyspermy (Table 1). When treatmenttime was 4.0 minutes, ability of the sperm to penetrate the eggs dropped greatly, and some eggsbecame polyspermic or failed to be activated by the sperm even though the sperm nuclei couldbe seen in their cytoplasm. Male gametes treated from 4.5 and 5.0 minutes fertilized only about10% of the eggs, and failed to activate any. None of the oocytes exposed to sperm irradiated for15 minutes were fertilized, and none were activated by any breakdown products of the heavilytreated sperm present in the seawater in which they were held during irradiation.

Based on these initial observations, the cytological nature of the activation of oyster eggstreated with sperm irradiated from 2.0-3.5 minutes was examined in detail to determine if anyportion of this was gynogenetic. The 4.0 exposure was repeated to determine if results at thisexposure were consistent. Higher exposure levels, along with non-exposed sperm, were used ascontrols.

One out of the 65 control eggs examined was a spontaneous haploid with 2 polar bodies. The remainder of the control eggs developed normally with chromosome contributions fromboth the male and female gamete and two polar body nuclei.

Aliquots of the eggs were mixed with sperm treated with ultraviolet light at incrementsfrom 0.5 to 15.0 minutes. When fertilized with sperm treated for 0.5 minutes, development was normal in half the eggs, but the other half showed various chromosome abnormalities (asbreakage, fragments, bridges) resulting from genetic damage to the chromosomes of theirradiated sperm. Exposure of the sperm for 1.0 minute did not cause much difference in theseincidences.

An exposure of 1.5 minutes resulted in gynogenetic stimulation of about 50% of the eggsas evidenced by the haploid number of their chromosomes and the presence of 2 polar bodynuclei. The absence of one polar body nucleus in 1 egg indicated that diploidy wasspontaneously restored in 1 gynogenetic egg by fusion of the reduced number of femalechromosomes with the second polar body. A third of the haploid eggs had irregular chromosomenumbers and irregular mitoses in some early cleavage cells. This category of defect waspersistent and appeared in haploid eggs fertilized with sperm treated at all gynogeneticallyeffective levels of irradiation.

In eggs stimulated by the sperm exposed to 1.5 minutes, another third of the gynogenetichaploids contained some fragments of incompletely destroyed male chromosomes. Thiscategory of egg also occurred in samples fertilized with sperm irradiated 2.0 and 2.5 minutes. When sperm were treated 3.0 minutes, the proportion of eggs with chromosome fragments

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dropped and did not occur at all in eggs fertilized with sperm treated 3.5 minutes. However, itappeared again in those fertilized with sperm irradiated 4.0 minutes. The genetically inactivatedsperm nucleus was visible in many eggs as a pale-staining, compact nucleus.

Influence of High Pressure Treatment on Unfertilized and Cleaving Eggs

High pressure treatment of spawned unfertilized eggs of the oyster seems to cause thearrested metaphase I chromosomes (bivalents) of the female gamete to dissociate into metaphaseII-like clusters of 20 chromosomes. In some eggs, the chromosome number was reduced to thehaploid 10 and what would have been the polar body nucleus removed from the region of thepsuedo-Metaphase II configuration. Five-minute treatment times of 8,000 and 14,000 PSI causedmost eggs to advance to the Metaphase II-like configuration without any fertilization. Resultswere not fully consistent over trials at different PSI-time combinations. High pressure treatmentfor more than 5 minutes, however, seems only to scatter intact bivalents about the egg. It is notknown what, if any, portion of such eggs can complete meiosis and cleavage.

When early cleavage eggs are subjected to high PSI, the effect is to produce a colchicine-like metaphase arrest of the chromosomes. At extremely high PSI there was a slight amount ofchromosome breakage, pulverization, or scattering. Treatment of eggs at later cleavage stagesproduced the same results. High pressure clearly has a strong effect on the spindle in both themeiotic Metaphase I eggs, and in mitotic cleavage stages.

Androgenesis

When eggs were treated with ultraviolet for only 1 minute, about 60% of the eggsappeared to develop normally and on schedule with the full, normal complement of femalechromosomes. About 30% of the eggs gave evidence of breakage of the female’s chromosomesfrom the less than totally effective exposure. However, about 10% of the eggs began androgenicdevelopment. This is based on cytological examination of 90 eggs, 6 hours post-fertilization.

When eggs were exposed to ultraviolet for 3 minutes, none of them developed normallywith the normal complement of female chromosomes. About 30% contained partiallydeteriorated chromosomes of the female zygote. About 13% of the eggs began development,and in 5% this was progressing normally toward cleavage (based on 128 eggs examined 6 hourspost-fertilization). In these eggs, the full complement of chromosomes of the female seemed tohave been destroyed. After 5 minutes’ exposure of the eggs, the chromosome content of thefemale appeared to be effectively destroyed in most eggs and was visible as a pale, vacuolatednucleus without chromatic structure.

Eggs irradiated for 5 minutes tended to be polyspermic. Sometimes close to 100 spermpenetrated these eggs. The male nuclei developing from such fertilization were readilydiscernible in eggs fixed 1.5 hours after fertilization. However, 7 of 30 eggs scored (23%) werepenetrated by a single sperm, the developing nucleus of which was clearly visible. Two of these contained fragments of female chromosomes incompletely destroyed by prior irradiation of theegg.

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By 6 hours after fertilization, the sperm in almost all such eggs had developed elongated,pro-metaphase-like chromosomes. Polyspermic eggs often had 100's of such chromosomesscattered about their cytoplasm. Even so, 22% of the eggs were observed to be clearly initiatingandrogenetic development. About half of these either were in ana- or telophase or hadmetaphase chromosomes doubled for a mitotic division. Two of 6 cleavages observed werequite normal.

DISCUSSION

Cytogenetic analyses of eastern oyster eggs and embryos revealed that haploiddevelopment can be induced at frequencies greater than 50% when eggs are stimulated byirradiated sperm. Androgenetic development is induced with irradiated eggs and untreatedsperm, but at lower frequencies. High pressure treatment of unfertilized eggs can initiateresumption of the meiotic process, and pressure treatment of cleavages and eggs fertilized withirradiated or with untreated sperm results in polyploidization. If such embryos can continuedevelopment, possibilities for applications of chromosome engineering in shellfish may begreater than in finfish because the meiotic stage of ripe shellfish eggs (metaphase I) is earlierthan that of finfish (telophase II).

This first thorough cytological examination to be conducted on any eggs of anaquaculture species in the process of gynogenetic stimulation with irradiated sperm, raises thequestion whether the total destruction of the male complement of sperm is possible at levels ofirradiation which still leave the sperm capable of penetrating and stimulating the female gameteto develop. Any inferior performance of gynogenetic shellfish may be influenced then byfragment chromosomes from the male, or less likely, there may be a positive influence of persisting fragment chromosomes on viability. This study also shows that there is a high level ofmitotic instability intrinsically associated with the haploid state. This could be due to alteredratios of chromosome number to spindle mass. Androgenesis is stimulated in the oyster lessfrequently than gynogenesis. It still occurs though, at levels that make its further studyworthwhile.

Should the oyster eggs with bivalent chromosomes at metaphase I dissociated by highpressure be capable of reasonably normal development, every embryo should be an almost exactreplica of the mother (clones). This deserves further study. Natural parthenogenesis occurs insome invertebrate groups through suppression of Metaphase I. Possibly though oyster eggs,subjected to high pressures and the likely attendant delays in development would not remainviable.

Ultraviolet light seems to be more effective in destruction of the genetic material ofsperm without inactivating the sperm physiologically than does X-irradiation. In a fewgynogenetic eggs there must be spontaneous fusion of the reduced number of oocytechromosomes with the second polar body nucleus. However, in most instances, diploidy wouldprobably have to be restored with a chemical agent, heat or cold-shock, or high pressure as hasbeen done successfully in several fish.

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The experimental observations reported here, and also instances of spontaneousparthenogenesis in eastern oyster eggs, both suggest that shellfish as well as finfish affordopportunities for chromosome manipulations. Opportunities, of course, depend further on theability of such eggs to develop and to do so before the egg ages too much, and upon the ability ofthe oyster to tolerate genetic homozygosity. Bay scallops, as hermaphrodites, might be moreamenable to induction of cloning and transgenesis. At least some groups of finfish, as thesalmonids, have a tetraploid ancestry which probably predisposes them to tolerate gynogeneticmethods of development. Also, there are finfish with naturally occurring gynogenetic andparthenogenetic methods of production. Neither polyploidy nor gynogenesis is known to occurnaturally in any group of pelecypod mollusks, although several studies show that bivalves cantolerate induced polyploidy. The greater fecundity of pelecypod mollusks would afford someopportunities for recovery of such embryos even if their viability were much lower than that infish. All types of genetics - molecular, biochemical and cytogenetic could play a critical role inthe evaluation, development and improvement of resources, including transgenics or geneticallymodified organisms. Furthermore, incorporating molecular marker technologies and geneticmanipulation into breeding programs could increase the efficiency of selection.

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Table 1. Cytological examination of fertilization of American oyster eggs with UV-treated sperm

__________________________________________________________________________________________________ Fertilization Fertilization Polyspermy No. fertilizationMinutes of and activation no activation No. eggs % No. eggs %irradiation No. eggs % No. eggs %___________________________________________________________________________________________________

0 100 100 0 0 0 0 0 0

2.0, 2.5, 3.0, 3.5 100 100 0 0 0 0 0 0

4.0 38 66.7 1 1.8 2 3.5 16 28.1

4.5 0 0 6 12.0 0 0 44 88.0

5.0 0 0 7 14.0 0 0 43 86.0

15.0 0 0 0 0 0 0 50 100

______________________________________________________________________________________________________

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Table 2. Spindle disruptive effect of high pressure on early cleavage eggs of the American oyster

___________________________________________________________________________

PSI for 5 min Effects on mitotic apparatus___________________________________________________________________________

Control Normal array of normal mitosis

2,000 - 3,000 Slight to no discernible effect

6,000 - 8,000 All divisions in colchicine-like metaphase arrest

10,000* - 14,000* All divisions in colchicine-like metaphase arrest__________________________________________________________________________

*Some evidence for chromosome breakage, pulverization and scattering - negligible_____________________________________________________________________________

********************************************************______________________________________________________________________________Table 3. Spindle disruptive effect of high pressure on mid-cleavage eggs of the American oyster

______________________________________________________________________________

PSI for 7 min Effects of mitotic apparatus

______________________________________________________________________________

Control Normal array of normal mitosis

11,000* - 13,000* Most divisions in colchicine-like metaphase arrest

______________________________________________________________________________

* Some evidence for some irregular mitosis______________________________________________________________________________

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Examples of Crossover Events in the ChromosomesOf the Oyster, Crassostrea virginica

1a

Diakinesis

1b

Metaphase I

Figure 1.

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Figure 2. Varied outcomes of manipulating Meiosis I in spawned eggs of oysters and other shellfish.

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Chromosome Engineering Models Based On Oyster Data

Figure3. Schematic models of cytological phenomena occurring after manipulating Meiosis I following addition of either normal or genetically inactivated sperm. One cross-over event is represented.

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BIBLIOGRAPHY

Ahmed, M. 1973. Cytogenetics of oysters. Cytologia 38: 337-346.

Ahmed, M. 1975. Speciation in living oysters. Adv. Mar. Biol. 13, 357-397.

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