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

Trends in genetics of bivalve mollusks: A review

S. Stiles and J. Choromanski Abstract

Recent advances in genome mapping and the development of linkage maps, as well as the production of polyploids and aneuploids, in bivalve mollusks have led to a resurgence of interest in chromosome organization and manipulation. Knowledge of normal meiosis and baseline data on mitoses in these invertebrates can assist in such undertakings and serve to elucidate genome features and processes. Baseline data will be presented on meiosis and genetic manipulation such as polyploidy, aneuploidy, and tetraploid cloning in the eastern , Crassostrea virginica. For example, approximately 12% of eggs from mass-spawned populations of C. virginica 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 of oysters, which can serve as a reference frequency for current studies on aneuploidy in eastern oysters. Previous chromosome engineering efforts for the induction of cloning or polyploidy in 15 experiments with eastern oysters revealed that the ploidy level of early embryos developed from eggs treated with cytochalasin B, high pressure and/or exposed to irradiated sperm in general ranged from haploidy through pentaploidy. Outcomes depended on the female, experimental conditions, synchronous development and whether or not the sperm were genetically inactivated with irradiation. Triploidy occurred as high as 66%, but generally ranged from 3% to 38%. Some embryos were chromosomal mosaics or aneuploids. Implications for genetic manipulation, including transgenesis, in other bivalves such as bay will be discussed. Results should be considered with regard to efforts for rehabilitating or restocking bivalve populations.

Key Words: Chromosomes, polyploidy, aneuploidy, cloning, bivalve mollusks, genome mapping, 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: [email protected], [email protected]

1 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 also provide a basis for the application of modern genetics to biological, agricultural and aquaculture sciences, including biotechnological advances. For example, relationships are being explored for DNA replication, Mendelian ratios and Hardy-Weinberg equilibrium. Genomic sequence databases are also being explored, providing new insights into how genes function as well as characterization of transposable elements. In addition, an understanding of dimensions between DNA and chromosomes is becoming necessary. However, such relationships between physical and genetic distances of genes and their impact on genetic events, gene expression, and evolution have been little elucidated.

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

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

The purpose of this present review, primarily of chromosomal genetics using the eastern oyster (Crassostrea virginica) as a model, was to evaluate status and trends including genome mapping and, particularly, cytological responses of a bivalve to some common means for inducing gynogenesis, androgenesis and polyploidy. The value of genetic manipulations such as gynogenesis 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. In addition, production of successful gynogenetic progeny offers some special opportunities for studying the quantitative inheritance of economic traits.

2 Chromosome Genetics

One of the bases to a better understanding of these approaches involves chromosomal variation and polymorphisms or genomic processes in evolution or selection. Concurrent with advances in molecular genetics has come increased awareness and understanding of the role of chromosomes as a significant part of genomic research in any organism. Additionally, the advent 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 serve as 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 new importance. Early development of maps was difficult due to the paucity of marker loci and to genotype environmental interactions which can modify the expression of qualitative traits. In addition, map utility has often been constrained by the lack of linkages to economically important traits.

Genome Mapping

Genome maps are created from markers - DNA sequences in or near genes whose locations are known - and compared with the occurrence of favorable traits. If the markers and traits appear together more often than would occur by chance, the locations of the genes for the desirable trait are likely to be near the markers. Multiple genes usually govern a single trait of economic 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 they have the desired QTLs. If so, marker - assisted selection (MAS) enables the researchers to put these traits into new breeding programs much sooner than if they used trial and error breeding to identify organisms with good genes. Comprehensive genome analyses should entail analyzing genome rearrangements in the context of cytogenetics, molecular genetics, population biology and breeding projects.

Advances in the characterization of bivalve chromosomes can contribute to genome mapping. Early studies involved karyotyping and chromosome banding with some degree of success. 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 loci were met by problems from distortions of Mendelian segregation ratios, as reviewed by Launcey and Hedgecock (2001). Based on microsatellite loci, these investigators reported distorted segregation ratios in Pacific oyster families and estimated the genetic load. It was hypothesized that selection against recessive deleterious mutations at closely linked genes was responsible for non-Mendelian inheritance of markers, beginning in the juvenile stage. They concluded that such 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 fitness genes with a few markers in a small genome. The haploid number of chromosomes in all Crassostrea and Ostrea species studied thus far is 10.

3 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 the genome 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 material for selection. One important mechanism for the genome to change its content of genes rather than a combination of alleles is crossing-over, or when a recombination event occurs between two sites that are not homologous. Crossover events in meiosis can be observed in bivalves as demonstrated in the prometaphase I or diakinesis group and metaphase I bivalents in Figures 1a and 1b.

Chromosome Manipulation

The development of triploid and tetraploid organisms has been a significant component of agricultural potential to enhance aquacultured species. While progress has been made in the area of chromosome manipulation, consistent results have not always been achieved. Basic approaches in bivalves include induction of triploidy, tetraploidy, gynogenesis, androgenesis and cloning.

Chromosome manipulation or engineering could play a significant role in the aquaculture of bivalves. Products could range from gynogens or clones of organisms with outstanding traits to polyploids which also could be commercially valuable. Gynogens could be produced in bivalve mollusks by stimulation of the egg to proceed through meiosis without fusion with the sperm or male contribution. Variation on the process could involve destruction of the oocyte nucleus 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 the contribution of either sex. Doubling of the haploid chromosomes then would result in a diploid gynogen, or the equivalent of a clone of the female or male exempt crossing over.

Unlike eggs of finfish, shellfish eggs divide holoblastically, therefore, their immediate post-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 the spawned, unfertilized shellfish oocyte is blocked at Metaphase I of meiosis, not Telophase II as in finfish, it affords opportunities to study and to manipulate even earlier meiotic stages than possible in finfish.

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

4 Regular Meiosis and Fertilization in Spawned Eggs of the Eastern Oyster

Normal, unfertilized, spawned eggs of C. virginica are either at prometaphase or metaphase 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. Oyster chromosomes consist of 10 homomorphic isopyknotic pairs. The genes occur linked in 10 different groups. Meiotic chromosomes have some chiasmata, an indication of crossing over of genes. Therefore, linkage of the oyster’s genes is not complete. Meiosis of the egg generally is of the usual type with genetic segregation. Similarly, meiotic divisions in the male gonad appear to 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 were not infrequent in the material used in these studies. Aneuploid, haploid, triploid, and chromosomally mosaic embryos are the result of such disturbances. They no doubt lead to poor embryo 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 resolved into chromosomes in a single egg. These extra nuclear groups can either remain in situ in the cytoplasm or several of them may fuse with the female group with the resultant formation of a multipolar spindle.

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

5 Experimental Manipulation with X-Irradiation

Parthenogenesis

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

For the induction of parthenogenesis, spawned sperm in seawater was irradiated using a Siemens Stabilpan X-ray especially calibrated for the high doses necessary for the invertebrate sperm. Eleven doses, ranging from 10,000 R to 225,000 R, were delivered to the oyster sperm in initial efforts to determine which dose might best induce parthenogenesis. Cytogenetic criteria for 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 of stages of male development. Spawned, unfertilized eggs of C. virginica usually remain at Metaphase I or the stage just preceding the ones mentioned above. In full-sib crosses of the eastern 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% with 15,000 and 225,000 R to the spawned sperm, to 15% with 150,000 R. Spontaneous parthenogenesis averaged only 1% in the control in accordance with earlier measurements (Longwell and Stiles, 1968; Stiles and Longwell, 1973). Development at 10,000 R represented 66% true fertilization, with the X-irradiated sperm contributing severally damaged chromosomes to the zygote. Similarly, at 15,000 R, the sperm participated in fertilization, producing cleaving embryos in 19% of the eggs. Ninety-six percent of the eggs cleaved. At 15,000 R, as many as 13.6% 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%; at 175,000 R and 225,000 R, 2.9% and 2.8%. It is uncertain if any of the straight-hinge larvae were developed from parthenogenic eggs. Induction of successful gynogenesis in oysters with irradiated sperm does not appear to display a clear Hertwig effect, and may not be so readily obtainable as in fish. It needs further investigation to appraise its practicality. Spontaneous parthenogenesis 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 genetic inactivation of oyster sperm intended for parthenogenetic stimulation of oyster eggs. It seems that the lower doses of 10,000 R and 15,000 R could be eliminated from further testing since these doses allowed true fertilization to occur. There was a significant decrease in development of eggs to the straight-hinge larval stage at 20,000 R. This could indicate a point of maximum

6 development problems resulting from contributions of damaged male chromosomes to the eggs in true fertilization. Doses from about 30,000 R or even higher than 225,000 R should be further tested.

Complicating the determination here is a sperm dilution factor, which can vary from experiment to experiment. The small size of the oyster sperm and the relatively small number of its 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 31,500 R, compared to effective doses of irradiation as high as 264,000 R delivered to dry or concentrated sperm (Rugh, 1953). Irradiated sperm in an aqueous medium may produce more obscure effects because of some interaction between products of irradiation and the medium alone.

Manipulating Division of Meiosis I to Clone Maternal Genomes of Shellfish

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

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

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

Genetic manipulations of chromosomes in bivalve shellfish generally have concerned induction of either gynogenesis for developing genetically homozygous lines or of polyploidy for obtaining sterile triploid strains. However, manipulation of meiosis I in bivalves, in contrast to manipulation of meiosis II in finfish (Streisinger et al., 1981), could also result in heterozygous genotypic copies of selected superior shellfish. This can be accomplished by induction of gynogenesis and suppression of meiotic divisions. The focus in this case is not increased homozygosity as selfing connotes, but the production of copies of a superior maternal genotype in the heterozygous state when maternal and paternal homologues are retained as they are found in the sole female parent. This outcome can likely occur in all organisms such as an oyster in which eggs are available in metaphase I of meiosis and therefore for suppression of

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

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

Gynogenesis and parthenogenesis have already been investigated in bivalves with some success 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, induction of 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 combined and effects assessed cytologically. The prime purpose was to identify and quantify those events which could lead to production of near identical maternal genomes or clones. Another purpose was to elucidate cytological events which must occur in procedures used with varying success to produce triploid shellfish.

Treatments for Inducing Gynogenesis and Polyploidy

Ultraviolet radiation of sperm for the purpose of its genetic inactivation was conducted with a short-wave UV lamp (254 nanometers) of 8 watts contained in a quartz envelope. Sperm suspensions of 50 ml were held for treatment in glass Petri dishes placed 15 cm from the light source. The concentration of sperm as measured by a hemocytometer was 4.2 X 106 per ml in seawater. Sperm exposed for 3.5 minutes had the greatest efficiency in inducing gynogenesis to eggs. 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 of seawater. This density was reduced to 750 eggs/ml when the first sample was taken for cytology and 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 was maintained at a usual 30 eggs/ml.

Eggs subjected to pressure treatments were held in small compartmentalized steel containers placed inside an alloy steel pressure vessel within 1-5 minutes after the addition of the sperm. 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 eggs were exposed for approximately 15 minutes to cytochalasin B at a concentration of 0.5 mg/l in

8 0.005% dimethyl sulfoxide (DMSO) solution. Ploidy levels of eggs, embryos, and larvae sampled at various times during development were determined by their direct cytological examination.

Induced Gynogenesis

Irradiation of oyster sperm for 2.0, 2.5, 3.0 and 3.5 minutes had no effect on its ability to fertilize oyster eggs, and the treatment did not cause polyspermy (Table 1). When treatment time was 4.0 minutes, ability of the sperm to penetrate the eggs dropped greatly, and some eggs became polyspermic or failed to be activated by the sperm even though the sperm nuclei could be seen in their cytoplasm. Male gametes treated from 4.5 and 5.0 minutes fertilized only about 10% of the eggs, and failed to activate any. None of the oocytes exposed to sperm irradiated for 15 minutes were fertilized, and none were activated by any breakdown products of the heavily treated 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 eggs treated with sperm irradiated from 2.0-3.5 minutes was examined in detail to determine if any portion of this was gynogenetic. The 4.0 exposure was repeated to determine if results at this exposure were consistent. Higher exposure levels, along with non-exposed sperm, were used as controls.

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 from both the male and female gamete and two polar body nuclei.

Aliquots of the eggs were mixed with sperm treated with ultraviolet light at increments from 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 (as breakage, fragments, bridges) resulting from genetic damage to the chromosomes of the irradiated sperm. Exposure of the sperm for 1.0 minute did not cause much difference in these incidences.

An exposure of 1.5 minutes resulted in gynogenetic stimulation of about 50% of the eggs as evidenced by the haploid number of their chromosomes and the presence of 2 polar body nuclei. The absence of one polar body nucleus in 1 egg indicated that diploidy was spontaneously restored in 1 gynogenetic egg by fusion of the reduced number of female chromosomes with the second polar body. A third of the haploid eggs had irregular chromosome numbers and irregular mitoses in some early cleavage cells. This category of defect was persistent and appeared in haploid eggs fertilized with sperm treated at all gynogenetically effective levels of irradiation.

In eggs stimulated by the sperm exposed to 1.5 minutes, another third of the gynogenetic haploids contained some fragments of incompletely destroyed male chromosomes. This category 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

9 dropped and did not occur at all in eggs fertilized with sperm treated 3.5 minutes. However, it appeared again in those fertilized with sperm irradiated 4.0 minutes. The genetically inactivated sperm 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 the arrested metaphase I chromosomes (bivalents) of the female gamete to dissociate into metaphase II-like clusters of 20 chromosomes. In some eggs, the chromosome number was reduced to the haploid 10 and what would have been the polar body nucleus removed from the region of the psuedo-Metaphase II configuration. Five-minute treatment times of 8,000 and 14,000 PSI caused most eggs to advance to the Metaphase II-like configuration without any fertilization. Results were not fully consistent over trials at different PSI-time combinations. High pressure treatment for more than 5 minutes, however, seems only to scatter intact bivalents about the egg. It is not known 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 of chromosome breakage, pulverization, or scattering. Treatment of eggs at later cleavage stages produced the same results. High pressure clearly has a strong effect on the spindle in both the meiotic Metaphase I eggs, and in mitotic cleavage stages.

Androgenesis

When eggs were treated with ultraviolet for only 1 minute, about 60% of the eggs appeared to develop normally and on schedule with the full, normal complement of female chromosomes. About 30% of the eggs gave evidence of breakage of the female’s chromosomes from the less than totally effective exposure. However, about 10% of the eggs began androgenic development. 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 normally with the normal complement of female chromosomes. About 30% contained partially deteriorated 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 hours post-fertilization). In these eggs, the full complement of chromosomes of the female seemed to have been destroyed. After 5 minutes’ exposure of the eggs, the chromosome content of the female appeared to be effectively destroyed in most eggs and was visible as a pale, vacuolated nucleus without chromatic structure.

Eggs irradiated for 5 minutes tended to be polyspermic. Sometimes close to 100 sperm penetrated these eggs. The male nuclei developing from such fertilization were readily discernible in eggs fixed 1.5 hours after fertilization. However, 7 of 30 eggs scored (23%) were penetrated 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 the egg.

10 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 chromosomes scattered about their cytoplasm. Even so, 22% of the eggs were observed to be clearly initiating androgenetic development. About half of these either were in ana- or telophase or had metaphase chromosomes doubled for a mitotic division. Two of 6 cleavages observed were quite normal.

DISCUSSION

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

This first thorough cytological examination to be conducted on any eggs of an aquaculture species in the process of gynogenetic stimulation with irradiated sperm, raises the question whether the total destruction of the male complement of sperm is possible at levels of irradiation which still leave the sperm capable of penetrating and stimulating the female gamete to develop. Any inferior performance of gynogenetic shellfish may be influenced then by fragment 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 of mitotic instability intrinsically associated with the haploid state. This could be due to altered ratios of chromosome number to spindle mass. Androgenesis is stimulated in the oyster less frequently than gynogenesis. It still occurs though, at levels that make its further study worthwhile.

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

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

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

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

______Fertilization Fertilization Polyspermy No. fertilization Minutes 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

______

13 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 ______

14 Figure 1.

Examples of Crossover Events in the Chromosomes Of the Oyster, Crassostrea virginica

1a

Diakinesis

1b

Metaphase I

15 Figure 2.

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

16 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.

Chromosome Engineering Models Based On Oyster Data

17 BIBLIOGRAPHY

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