International Council
for the Exploration of the Sea
C.M. 2002/U:11
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 oyster, Crassostrea
virginica. 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 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 scallops 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 postfertilization 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 510 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 colchicinelike 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
2.0, 2.5, 3.0, 3.5
100
4.0
100
0
0
0
0
0
0
100
0
0
0
0
0
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
Ahmed, M. 1973. Cytogenetics of oysters. Cytologia 38: 337-346.
Ahmed, M. 1975. Speciation in living oysters. Adv. Mar. Biol. 13, 357-397.
Ahmed, M. and A.K. Sparks. 1967. A preliminary study of chromosomes of two species of
oyster (Ostrea lurida and Crassostrea gigas). J. Fish. Res. Board Can. 24: 2155-2159.
Ahmed, M. & Sparks, A.K. 1970. Chromosome number, structure and autosomal
polymorphism in the marine mussels Mytilus edulis and Mytilus californianus. Biol.
Bull. 138, 1-13.
Allen, R.D. 1953. Fertilization and artificial activation in the egg of the surf clam,
Spisula solidissima. Biol. Bull., 105: 213-239.
Allen, S.K., Jr., Gagnon, P.S. & Hidu, H. 1982. Induced triploidy in the soft-shell clam. J.
Hered. 73, 421-428.
Allen, S.K., Jr. 1983. Flow cytometry: assaying experimental polyploid fish and shellfish.
Aquaculture 33: 317-328.
Allen, S.K., Jr. 1987. Gametogenesis in three species of triploid shellfish: Mya arenaria,
Crassostrea gigas and Crassostrea virginica. Proc. World Symp. on Selection,
Hybridization, and Genetic Engineering in Aquaculture. Vol. II. Berlin 1987. Bordeaux,
May 27-30, 1986.
Allen, S.K. Jr., S.L. Downing and K.K. Chew. 1989. Hatchery Manual for Producing Triploid
Oysters. Seattle, WA: University of Washington Press, Seattle, 27 pp.
Allen, S.K., Jr., and S.L. Downing. 1990. Performance of triploid Pacific oysters, Crassostrea
gigas; gametogenesis. J. Fish. Aquat. Sci. 4: 1214-1222.
Allen, S.K. Jr. and P.M. Gaffney. 1993. Genetic confirmation of hybridization between
Crassostrea gigas (Thunberg) and Crassostrea rivularis (Gould). Aquaculture 113:
291-300.
Allen, S.K. Jr., P.M. Gaffney, J. Scarpa and B. Bushek. 1993. Inviable hybrids of Crassostrea
virginica (Gmelin) with C. rivalus (Gould) and C. gigas (Thunberg). Aquaculture 113:
269-289.
Allen, S.K. Jr., M. Shpigel, S. Utting and B. Spencer. 1994. Incidental production of tetraploid
Manila clams, Tapes phillippinarium (Adams and Reeve). Aquaculture 128: 13-19.
18
Arai, K., Naito, F., Sasaki, H. & Fujino, K. 1984. Gynogenesis with ultraviolet ray irradiated
sperm in the Pacific abalone. Bull. Jpn. Soc. Sci. Fish. 50, 2019-2023.
Barber, B.J., R. Mann and S.K. Allen Jr. 1992. Optimization of triploid induction for the oyster
Crassostrea virginica Gmelin. Aquaculture 106: 21-26.
Beaumont, A.R. and J.E. Fairbrother. 1991. Ploidy manipulation in molluscan shellfish. A.
review. J. Shellfish Res. 10: 1-18.
Bennett, M.D., Bobrown, M. & Hewitt, G. 1981. Chromosomes Today, Vol. 7. George Allen
and Unwin, London
Bickham, J.W., Tucker, P.K. & Legler, J.M. 1985. Diploid-triploid mosaicism: an unusual
phenomenon in side-necked turtles (Platemys platycephala). Science 227, 1591-1593.
Brown, M.V., L. Strausbaugh and S. Stiles. 2000. Methodology for the generation of molecular
tags in Placopecten magellanicus (sea scallop) and Argopecten irradians (bay scallop).
J. Shellfish Research. Vol. 19(1): p. 569.
Brutlag, D.L. 1980. Molecular arrangement and evolution of heterochromatic DNA. Ann. Rev.
Genet. 14, 121-144.
Buroker, N.E., W.K. Hershberger & K.K. Chew. 1979. Population genetics of the family
Ostreidae. II. Interspecific studies of the genera Crassostrea and Saccostrea. Mar. Biol.
54: 171-184.
Carson, H.L. 1967. Selection for parthenogenesis in Drosophila mercatorum. Genetics 55, 157171.
Chaiton, J.A. and S.K. Allen, Jr. 1985. Early detection of triploidy in the larvae of Pacific
oysters, Crassostrea gigas, by flow cytometry. Aquaculture 48: 35-43.
Choromanski, J., S. Stiles, C. Cooper, E. Beden, S. Lonergan and P. Trupp. 1999. Growth and
survival of bay scallops from genetic lines at different densities and depths: Collaborative
study between the NMFS and Bridgeport Aquaculture School. J. Shellfish Research 18
(1): 263.
Chourrout, D. 1983. Pressure-induced retention of second polar body and suppression of first
cleavage in rainbow trout: production of all-triploids, all-tetraploids, and heterozygous
and homozygous diploid gynogenetics. Aquaculture 36: 111-126.
Chun, C.Z., S. Seri, S. Stiles and T.T. Chen. 2002. Production of transgenic mollusks and
crustaceans. J. Shellfish Research. Vol. 19(1): p. 576.
19
Chur, E.H.Y., H.C. Thuline and D.E. Norby. 1964. Triploid-diploid chimerism in a male
tortoiseshell cat. Cytogenetics 3: 1-18.
Comings, D.E. 1978. Mechanisms of chromosome banding and implications for chromosome
structure. L Ann. Rev. Genet. 12: 25-46.
Cox, E.S., M.S.R. Smith, J.A. Nell and G.B. Maquire. 1996. Studies on triploid oysters in
Australia. VI. Gonad development in diploid and triploid Sydney rock oyster Saccostrea
commercialis (Iredale and Roughley). J. Exp. Mar. Biol. Ecol. 197: 101-120.
Cuellar, O. 1974. On the origin of parthenogenesis in vertebrates: the cytogenetic factors.
Amer. Natur. 108: 625-648.
Davis, H.C. 1950. On interspecific hybridization in Ostrea. Science 111: 522.
Dawson, G.W.P. 1962. An Introduction to the Cytogenetics of Polyploids. Blackwell Scientific
Publications. Oxford. 91 pp.
Downing , S.L. and S.K. Allen, Jr. 1984. Induced triploidy in the Pacific oyster, Crassostrea
gigas, optimal treatments with cytochalasin B depend on temperature. Aquaculture 61:
1-15.
Downing, S.L. 1988. Comparing adult performance of diploid and triploid monospecific and
interspecific Crassostrea hybrids. J. Shellfish Res. 7: 549 (abstract).
Downing, S.L. 1991. Hybrids among the Pacific, American and the Suminoe oysters: induction
and aquaculture potential (Abstract). J. Shellfish Res. 10: 514.
Durán-González, A., Rodríguez-Romero, F. & Laguarda-Figueras, A. 1984. Polymorphisme
chromosomique et nombre diploide dans une population d’Isognomon alatus (Bivalvia:
Isognomonidaw). Mal. Rev. 17, 85-92.
Eversole, A.G., C.J. Kempton, N.H. Hadley and W.R. Buzzi. 1996. Comparison of growth,
survival, and reproductive success of diploid and triploid Mercenaria mercenaria. J.
Shellfish Res. 15: 689-694.
Fairbrother, J.E. 1994. Viable gynogenetic diploid Mytilus edulis (L.) Larvae produced by
ultraviolet light irradiation and cytochalasin B. shock. Aquaculture126: 25-34.
Farinelli-Ferruzza, N. and Vitturi, R. 1981. Induction of triploids by hydrostatic pressure in the
species Ascidiella aspersa (Ascidiacea). J. Embryol. Exper. Morphol. 2, 155-162.
Foresti, F., Almeida Toledo L.F. & Toledo, S.A.F. 1981. Polymorphic nature of nucleolus
organizer regions in fish. Cytogenet. Cell Genet. 31, 127-144.
20
Gaffney, P.M. and S.K. Allen, Jr. 1993. Hybridization among Crassostrea species: A review.
Aquaculture 116: 1-3.
Gall, J.G. & Pardue, M.L. 1971. Nucleic acid hybridization in cytological preparations. Meth.
Enzymol. 21, 470-480.
Gendrau, S. and H. Grizel. 1990. Induced triploidy and tetraploidy in the European flat oyster,
Ostrea edulis L. Aquaculture 90: 229-238.
Gillespie, L.L. & Armstrong, J.B. 1979. Production of triploid and gynogenetic diploid axolotls
(Ambystoma mexicanum) by hydrostatic pressure. J. Exper. Zool. 210, 117-122.
Goldberg, R.B. and 9 co-authors. 1975. DNA sequence organization in the genome of five
marine invertebrates. Chromosoma 51: 225-251.
Goldberg, R.B., Crain, W.R., Ruderman, J.V., Moore, G.P., Barnett, T.R., Higgins, R.C.,
Gelfand, R.A., Galau, G.A., Britten, R.J. & Davidson, E.H. 1975. DNA sequence
organization in the genomes of five marine invertebrates. Chromosoma 51, 225-251.
Graham, J. & Taylor, C.J. 1980. Automated chromosome analysis using the Magicscan Image
Analyzer. Anal. Quant. Cytol. 4, 237-242.
Guo, X. 1989. Aneuploid Pacific oyster larvae produced by treating with cytochalasin B during
meiosis 1 (Abstract). J. Shellfish Res. 8: 445.
Guo, X., K. Cooper, W.K. Hershberger and K. Chew, 1991. Production of tetraploid embryos in
the Pacific oyster, Crassostrea gigas: comparison among different approaches (Abstract).
J. Shellfish Res. 10: 236.
Guo, X., W.K. Hershberger, K. Cooper and K.K. Chew. 1992. Genetic consequences of
blocking polar body 1 with cytochalasin B in fertilized eggs of the Pacific oyster,
Crassostrea gigas II: Segregation of chromosomes. Biol. Bull. 183: 387-393.
Guo, X., W.K. Hershberger, K. Cooper and K.K. Chew. 1993. Artificial gynogenesis with
ultraviolet light-irradiated sperm in the Pacific oyster, Crassostrea gigas. I. Induction
and survival. Aquaculture 113: 201-214.
Guo, X. and S.K. Allen, Jr. 1994a. Reproductive potential and genetics of triploid Pacific
oysters Crassostrea gigas (Thunberg). Biol. Bull. 187: 309-318.
Guo, X. and S.K. Allen, Jr. 1994b. Viable tetraploids in the Pacific oyster (Crassostrea gigas
Thunberg) produced by inhibiting polar body 1 in eggs from triploids. Mol. Mar.
Biotechnology 3: 42-50.
21
Guo, X., G.A. DeBrosse and S.K. Allen, Jr. 1996. All-triploid Pacific oysters (Crassostrea gigas
Thunberg) produced by mating tetraploids and diploids. Aquaculture 142: 149-161.
Hirschfeld, B., A.K. Dhar, K. Rask and A. Alcivoar-Warren. 1999. Genetic diversity in the
eastern oyster (Crassostrea virginica) from Massachusetts using the RAPD technique.
J. Shellfish Res., Vol. 18, No. 1, 121-125.
Hoskins, M.M. 1918. Further experiments on the effect of heat on the eggs of Cumingia. Biol.
Bull., 35: 260-272.
Huvet, A., K. Balabaud, N. Bierne, and P. Boudry. 2001. Microsatellite analysis of 6-hour-old
embryos reveals no preferential intraspecific fertilization between cupped oysters
Crassostrea gigas and Crassostrea angulata. Mar. Biotechnol. 3, 448-453.
Huvet, A., S. Lapégue, A. Magoulas, and P. Boudry. Mitochondrial and nuclear DNA
phylogeography of Crassostrea angulata, the Portuguese oyster endangered in Europe.
Conservation Genetics 1: 251-260.
Imai, T., and S. Sakai. 1961. Study of breeding of Japanese oysters, Crassostrea gigas. Tohoku
Journal of Agricultural Research 12: 125-171.
Ieyama, H. 1975. Chromosomes of the oysters Hyotissa imbricata and Dendostrea folium
(Bivalvia: Pteriomorphia). Venus 49: 63-68.
Inaba, F. 1936. Studies on artificial parthenogenesis of Ostrea gigas Thunberg. J. Sci.
Hiroshima U., Ser. B, Div. 1, Vol. 5, Art. 2: 29-46.
Kamalay, J.C., Ruderman, J.V., and Goldberg, R.B. 1976. DNA sequence repetition in the
genome of the American oyster. Biochem. Biophys. Acta 432: 121-128.
Kidder, G. 1976a. The ribosomal RNA cistrons in clam gametes. Dev. Biol. 49, 132-142.
Kidder. G. 1976b. RNA synthesis and the ribosomal cistrons in early molluscan development.
Ann. Zool. 16, 501-520.
Komaru, A. and K.T. Wada. 1994. Meiotic maturation and progeny of oocytes from triploid
Japanese oyster (Pinctada fucata martensii) fertilized with spermatozoa from diploids.
Aquaculture 120: 61-70.
Komaru, A., J. Scarpa and K.T. Wada. 1994.Ultrastructure of spermatozoa from induced
triploid Pacific oyster, Crassostrea gigas. Aquaculture 123: 217-222.
Komaru, A., J. Scarpa and K.T. Wada. 1995. Ultrastructure of spermatozoa in induced
tetraploid mussel Mytilus galloprovincialis (LMK). J. Shellfish Res. 14: 405-410.
22
Lapégue, S., I. Boutet, A. Leit|o, S. Heurtebise, P. Garcia, C. Thiriot-Quiévreux and P. Boudry.
2002. Biol. Bull. 202: 232-242.
Launcey, Sophie and Dennis Hedgecock. 2001. High genetic load in the Pacific oyster
Crassostrea gigas. Genetics 159: 255-265
Leitâo, Boudry, P., McCombie, H., Gerard, A., Thiriot-Quiévreau. 2002. Experimental
evidence for a genetic basis to differences in aneuploidy in the Pacific oyster (Crassostrea
gigas). Aquat. Living Resour. 14: 233-237.
Lewis, W.H. (Ed.). 1980. Polyploidy: Biological Relevance, Plenum, New York. 583 pp.
Lima-de-Faria, A. 1983. Molecular Evolution and Organization of the Chromosome. Elsevier,
Amsterdam.
Longo, F.J., 1972. The effects of cytochalasin B on the events of fertilization in the surf clam,
Spisula solidissima, polar body formation. J. Exp. Zol., 182: 321-344.
Longo, F.J. 1983. Meiotic maturation and fertilization. In: The Mollusca, Vol. 3, Development.
Academic Press, New York.
Longwell, A.C., S.S. Stiles and D.G. Smith. 1967. Chromosome complement of the American
oyster Crassostrea virginica, as seen in meiotic and cleaving eggs. Can. J. Genet. Cytol.,
9: 845-856.
Longwell, A. Crosby. 1968. Oyster genetics; research and commercial applications.
Conference on Shellfish Culture, April 1968, Suffolk Community College, Seldon, Long
Island, New York, p. 91-103.
Longwell, A.C. and S.S. Stiles. 1968a. Fertilization and completion of meiosis in spawned eggs
of the American oyster, Crassostrea virginica Gmelin. Caryologia 21: 65-73.
Longwell, A.C and S. Stiles. 1968b. Removal of yolk from oyster eggs by Soxhlet extraction
for clear chromosome preparations. Stain Technology, 43: 63-68.
Longwell, A. Crosby, and S.S. Stiles. 1973. Oyster genetics and the probable future role of
genetics in aquaculture. Malacological review 6(2): 151-177.
Longwell, A.C. and S. S. Stiles. 1973. Gamete cross incompatibility and inbreeding in the
commercial American oyster, Crassostrea virginica Gmelin, Cytologia, 38: 521-533.
23
Longwell, A.C. 1985. Subverting random segregation of genes to produce clones of superior
performing mollusks, fish or crustaceans. Intern. Council Explor. Sea, C.M. 1985/F:20
Mariculture Committee, Ref. Shellfish and Anadromous and Catadromous Fish
Committees. 24 pp.
Longwell, A.C. 1986. Critical review of methodology and potential of interspecific
hybridization. In: Tiews, editor. EIFAC/FAO Symposium on Selection, Hybridization
and Genetic Engineering in Aquaculture of Fish and Shellfish for Consumption and
Stocking. Berlin: Heenemann Verlogsgesellschaft, Vol. 2, 1987, pp. 3-21.
Longwell, A.C. 1987. Description of chromosomes and standard cytogenetic techniques for
wild and cultured shellfish molluscs. Pages 189-215 in Shellfish Culture, Development
and Management. Institut Francais de Recherches pour l”Exploitation de la mer,
LaRochelle.
Longwell, A. and S. Stiles. 1996. Chapter 12, “Chromosomes, Biology, and Breeding” in: The
Eastern Oyster Crassostrea virginica. V. Kennedy, R. Newell, and A. Eble (eds.). 734 pp.
Loosanoff, V.L. and H.C. Davis. 1963. Rearing of bivalve mollusks, p. 1-136. In: F.S. Russell
(ed.), Advances in Marine Biology. London, Academic Press.
Lyu, S. and S.K. Allen, Jr. 1999. Effect of sperm density on hybridization between Crassostrea
virginica (Gmelin) and C. gigas (Thunberg). J. Shellfish Res. 2: 459-464.
Maldonado-Amparo, Rosalío and Ana M. Ibarra. 2002. Ultrastructural characteristics of
spermatogenesis in diploid and triploid catarina scallop (Argopecten ventricosus
Sowerby II, 1842). J. Shellfish Res. Vol. 21, No. 1, 93-101.
Menzel, R.W. 1968. Cytotaxonomy of species of clams (Mercenaria) and oysters (Crassostrea).
Proc. Symp. on Mollusca, Part I, p. 75-84.
Morris, M. 1917. A cytological study of artificial parthenogenesis in Cumingia. J. Exp. Zool.,
22(1): 1-51.
Moynihan, E.P. and G.A.T. Mahon. 1983. Quantitative karotype analysis in the mussel Mytilus
edulis L. Aquaculture 33: 301-309.
Picozza, E., J. Crivello, M. Brown. L. Strausbaugh and S. Stiles. 2000. Status report for the
characterization of the Argopecten irradians genome. J. Shellfish Res., Vol. 19(1): p.
578.
Purdom, C.E. 1983. Genetic engineering by the manipulation of chromosomes. Aquaculture
33, 287-300.
24
Que, Huayong and Standish K. Allen, Jr. 2002. Hybridization of tetraploid and diploid
Crassostrea gigas (Thunberg) with diploid C. ariakensis (Fujita). J. Shellfish Res. Vol.
27, No. K. 137-143.
Rebordinos, L., P. Garcia and J.M. Cantoral. 1999. Founder effect, genetic variability, and
weight in the cultivated Portuguese oyster Crassostrea angulata. J. Shellfish Res., Vol.
18, No. 1, 147-153.
Rodríguez-Romero, F., M. Uribe-Alcocer, A. Laguarda-Figueras and Ma. E. Diupotex-Chong.
1979. The karotype of Crassostrea rhizophorae (Guilding, 1828). Venus 38: 135-140.
Rodríguez-Romero, A. Laguarda-Figueras, M. Uribe-Alcocer and M.L. Rojas-Lara. 1979.
Distribution of “G” bands in the karyotype of Crassostrea viginica. Venus 38: 180-184.
Rugh, R. 1953. X-irradiation of marine gametes. A study of the effcts of x-irradiation at
different levels on the germ cells of the clam, Spisula (formerly Mactra). Biol. Bull.,
104: 197-209.
Ruiz-Verdugo, C.A., J.L. Ramirez, S.K. Allen Jr. and A.M. Ibarra. 2000. Triploid catarina
scallop (Argopecten ventricosus Sowerby II, 1842): growth, gametogenesis, and
suppression of functional hermaphroditism. Aquaculture 186: 13-32.
Ruiz-Verdugo, C.A., S.K. Allen Jr. and A.M. Ibarra. 2001. Family differences in success of
triploid induction and effects of triploidy on fecundity of catarina scallop (Argopecten
ventricosus). Aquaculture 201: 19-33.
Scarpa, J. 1985. Experimental production of gynogenetic and parthenogenetic Mulinia lateralis
(Say). M.S. thesis, University of Delaware, Newark. 55 pp.
Scarpa, J., A. Komaru and K.T. Wada. 1994. Gynogenetic induction in the mussel, Mytilus
galloprovincialis. Bull. Natl. Res. Inst. Aquaculture 23: 33-41.
Scarpa, J., J.E. Toro and K.T. Wada. 1994. Direct comparison of six methods to induce
triploidy in bivalves. Aquaculture 119: 119-133.
Southworth, J., M. Brown, S. Stiles and L. Strausbaugh. 1999. Methodology for the generation
of polymorphic molecular tags in the bay scallop, Argopecten irradians. J. Shellfish
Research 18(1): 278.
Stanley, J.G., S.K. Allen, Jr. and H. Hidu. 1981. Polyploidy induced in the American oyster,
Crassostrea virginica, with cytochalasin B. Aquaculture 23: 1-10.
Stanley, J.G., H. Hidu and S.K. Allen, Jr. 1984. Growth of American oysters increased by
polyploidy induced by blocking meiosis I but not meiosis II. Aquaculture 37: 147-155.
25
Stephens, L.B. and S.L. Downing. 1988. Inhibiting first polar body formation in Crassostrea
gigas produces tetraploids, not meiotic triploids (Abstract). J. Shellfish Res. 7: 550.
Stiles, S.S. 1973. Cytogenetic analysis of an attempted interspecies hybridization of the oyster.
Incompatibility Newsletter 3: 41-45.
Stiles, S. and A. Crosby Longwell. 1973. Fertilization, meiosis and cleavage in eggs from large
mass spawnings of Crassostrea virginica Gmelin, the commercial American oyster.
Caryologia, 26(2): 253-262.
Stiles, S. 1978. Conventional and experimental approaches to hybridization and inbreeding
research in the oyster. In J.W. Avault, Jr. (ed.) Proc. 9th Annual Meeting World
Mariculture Society, 9: 577-586.
Stiles S. 1981. Recent progress on directed breeding experiments in Long Island Sound oysters.
Paper for Mariculture Committee, ICES, 1981 Stat. Meeting, Woods Hole, Mass. USA,
C.M. 1981/F:33 Maric. Comm. 14 p.
Stiles, S., J. Choromanski and A. Longwell. 1983. Cytological appraisal of prospects for
successful gynogenesis, parthenogenesis and androgenesis in the oyster. Intern. Council
Explor. Sea, C.M. 1983/F:10 Mariculture Committee, Ref. Shellfish Committee. 15 pp.
Stiles, S. and J. Choromanski. 1986. Manipulating meiosis I in shellfish to produce polyploids
and maternal clones (Abstract). Genetics 113, Supplement I (2), 10.4.
Stiles, S. and J. Choromanski. 1987. A method for cytogenetic and cytological examination of
small shelled larvae of bivalves and other zooplankton. Stain Technology 62(2).
113-118.
Stiles, S.S. and J. Choromanski. 1990. A genetic model of polyploidy and hybridization in
shellfish (Abstract). Environmental Auditor 2:8.
Stiles, S. and J. Choromanski. 1994. Prospects for the genetic improvement of shellfish in Long
Island Sound. Aquaculture Seminar, Milford, CT. J. Shellfish Res. 13(1): 120
Stiles, S. and J. Choromanski. 1995. Inbreeding studies on the bay scallop, Argopecten
irradians. J. Shellfish Res. 14(1): 278, Abstract.
Stiles, S., J. Choromanski and C. Cooper. 1998. Selection studies on growth and survival of bay
scallops, (Argopecten irradians) from Long Island Sound. J. Shellfish Res. 17(1): 363,
Abstract.
Stiles, S., J. Choromanski, C. Cooper and Q-Z Xue. 1998. Genetic and breeding investigations
of bay scallops (Argopecten irradians). 18th International Congress of Genetics
Proceedings. Beijing, China 8P218. P. 173.
26
Streisinger, G., Walker C., Dower N., Knauber D. & Singer F. (1981). Production of clones of
homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293-296.
Swarup, H. 1959. Effect of triploidy on the body size, general organization and cellular
structure in Gasterosteus aculeatus (L). J. Genet. 56: 143-155.
Tabarini, C.L. 1984. Induced triploidy in the bay scallop, Argopecten irradians and its effects
on growth and gametogenesis. Aquaculture 42, 151-160.
Thiriot-Quièvreux, C. and N. Ayraud. 1982. Les caryotypes de quelques espèces de bivalves et
de gastèropodes marins. Mar. Biol. 70: 165-172.
Thiriot-Quièvreux, C. 1984. Analyse comparè des caryotypes d”Ostreidae (Bivalvia). Cah.
Biol. Mar. 25: 407-418.
Thiriot-Quièvreux, C. and N. Ayraud. 1984. Chromosome analysis of three species of Mytilus
(Bivalve: Mytilidae). Mar. Biol. Letters 5, 265-273.
Thiriot-Quièvreux, C., T. Noël, S. Bougrier and S. Dallot. 1988. Relationships between
anueploidy and growth rate in pair matings of the oyster Crassostrea gigas. Aquaculture
75: 89-96.
Thiriot-Quièvreux, C. and A. Insua. 1992. Nucleolar organizer region variation in the
chromosomes of three oyster species. J. Exp. Mar. Biol. Ecol. 157: 33-40.
Thorgaard, G.H. and S.K. Allen, Jr. 1986. Chromosome manipulation and markers in fishery
management: In: N. Ryman and F.M. Utter, editors. Population genetics and its
application to fisheries management, Seattle: University of Washington Sea Grant, pp.
319-331.
Van der Meer, J. and M. U. Patwary. 1983. Genetic modification of Gracilaria tikvahiae
(Rhodophyceae). The production and evaluation of polyploids. Aquaculture 33: 311316.
Widman, J., J. Choromanski, R. Robohm, S. Stiles, G. Wikfors, A. Calabrese. 2001. A Manual
for Hatchery Culture of the Bay Scallop Argopecten irradians. CT Sea Grant (N.
Balcom, ed.). 49 pp.
Xue, Qin-Zhao, S. Stiles, and J. Choromanski, 1995. A Population Genetics Study of the Bay
Scallop, Argopecten irradians. A Report on the Joint US-China population genetics
research. 10 pp.
Yamamoto, S. and Y. Sugawara. 1988. Induced triploidy in the mussel Mytilus edulis by
temperature shock. Aquaculture 72: 21-29.
27
Zhang, Q., G. Yu, R.K. Cooper and T.R. Tiersch. 1999. High-resolution analysis of karyotypes
prepared from different tissues of the eastern oyster Crassostrea virginica. J. Shellfish
Res., 18(1): 115-120.
Zouros, F. 1988. Heterozygote superiority and genetic load for growth in natural populations of
marine bivalves (Abstract). J. Shellfish Res. 7: 181.
28