This paper not to be cited without prior reference to the author

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1 This paper not to be cited without prior reference to the author International Council for the Exploration of the Sea C.M.1983/F: 10 Mariculture Cttee Ref. Shellfish Cttee e CYTOLOGICAL APPRAISAL OF PROSPECTS FOR SUCCESSFUL GYNOGENESIS, PARTHENOGENESIS AND ANDROGENESIS IN THE OYSTER S. Stiles, J. Choromanski and A. Longwell National Marine Fisheries Service Northeast Fisheries Center Milford Laboratory Milford, Connecticut e.

2 .. ABSTRACT Cytogenetic analyses of American oyster eggs and embryos revealed that haploid development can be induced at frequenc~es 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 11).... "> I \ Les analyses cytogenetiques des oeufs et des embryons de 1 Ihultre americaine revelaient que le developpement "haploid" peut etre produit aux frequences plus" de 50% quand le sperme irradie fertilisait les oeufs. Le de~eloppement "androgenetic" est provoque par les oeufs irradies et le sperme sans traitement, mais aux frequences inferieures. Le traitement des oeufs infertiiises a haut pression peut causer la reprise du processus "miotic", et le traitement de la pression des divisions et des oeufs fertilises, avec le sperme irradie ou avec le sperme sans traitement, se termine par "polyploidization". Si tels embryons peuvent continuer leur developpement, les possibilites des applications de 1 'ingenieur chromosome seraient plus eleves au coquillage "qu1au poisson parce-que le stade "miotic" des oeufs murs du coquillage ("Hetaphase 1") vient plus tot que celui du poisson ("Telophase II")

3 . ~ - INTRODUCTION Unlike eggs of finfish, shellfish eggs divide holoblastically; therefore, their.. t I'. immediate post-fertilization chromosome stages can be studied with considerable ease... ' '. l. and very reliably (Longwell et ~., Longwell,1973)...,. 1967; Longwell and Stiles, 1968a; Stilesand Because the spawned, unfertilized shellfish oocyte is blocked at I Metaphase I of meiosis, not Telophase 11 as in finfish, it affords opportunities :. ~ to study and to manipulate even earlier meiotic stages than possible in finfish.... The purpose of this present study is to evaluate cytologically the response i,. /. i. of the American oyster (Crassostrea virginica) egg to some common means for inducing' ~,. j..",... gynogenesis, androgenesis and polyploidy. The value of gynogenesis in practical breeding,. ".;. \.. and in basic research has been pointed out by Purdom (1983), Kirpichnikov (1981), and!. I,.'. Stanley (1974). In addition, production of successful gynogenetic progeny offers some special opportunities for studying quantitative inheritance of economic traits. Materials and Methods Oysters from Long Island Sound provided eggs and sperm used for the experiments,.' described here.....,, " in separate containers. Spawning was induced soley by thermal stimulation of single oysters t '.,I, '.,, Ultraviolet radiation of the oocytes and sperm was accomplished with a short-wave UV lamp (254 nanometer~) of 8 watts contai~ed in a ~uart~ envelope. Seawater suspensio~ of 50 ml of sperm or oocytes were held for treatment in 'glass Petri dishes approxim~tely.. \ I 15 cm from the light source. Cell suspensions were 10 mm high in the dish. "'\ High pressure treatments were administered to eggs-in an alloy steel pressure ",', ). vessel. Eggs were held for treatment in small compartmentalized steel containers of su itab1e mesh. Late meiotic and cleavage stage eggs were fixed in alcohol:acetic acid (3:1) for cytological study. When necessary, their yolk content was removed in a Soxhlet apparatus using methano1:ch1oroform, 1:1 (Longwe11 and Stiles, 1968b). Squash preparations were made in 2% areto-orcein with a few ml of proprionic acid and eggs j

4 then examined with high-power phase contrast microscopy. Induced Gynogenesis Irradiation of oyster sperm for 2.0,2.5, 3.0 and 3.5 minutes had no effect on its ability to ferti1izeoyster eggs, and treatment did not cause po1yspermy (Tab1e 1). When treatment time was 4.0 minutes, ability of the sperm to penetrate the eggs droppedgreat1y, and so~e eggs became po1yspermic or fai1ed to be activated by the sperm even thoughthe sperm nuc1ei cou1d be seen in their cytop1asm. Male gametes treated for 4.5 and 5.0 mnutes ferti1izedon1y about 10% of the eggs, and fai1ed to activate any~ None of the oocytes exposed to sperm irradiated for 15 minutes were ferti1ized, and none were activated by any breakdown products of the heavi1y treated sperm present in the seawater in which they were held during irradiation. Based on these initial observations, the cyto10gica1 nature of the activation of oyster eggs treated with sperm irradiated from minutes was examined in detail to determine if any portion of this was gynogenetic. repeated to determine if resu1ts at this exposure were consistent. The 4.0 exposure was levels, a10ng with non-exposed sperm, were used as contro1s. See Tab1e 2. Higher exposure As shown in Tab1e 2,'1 out of the 65 contro1 eggs examined was' a spontaneous. haploid with 2'po1ar bodies. The remainder of the contro1 eggs deve10ped norma11y with chromosome contributions from both the male and fema1e gamete and two polar body nuc1e{~ A1iquots ofthe eggs were mixed with sperm treated with u1travio1et light at increments from 0.5 to 15.0 minutes. When ferti1ized with sperm treated for 0.5 minutes, deve10pment was normal in half the eggs, but the other half showed various chromosome abnorma1ities (as breakage, fragments, bridges) resu1ting 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 resu1ted ingynogenetic' stimulation of about 50% of the eggs as evidenced by the haploid number of their chromosomes and the presence of - 3 -

5 2 polar body nuclei. The absence of one polar body.nucleusin 1 egg indicated that.diploidy was spontaneously re~tored.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 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 fertil'ized with sperm irradiated 2.0 and 2.5 minutes. When sperm were treated 3.0 minutes; the proportion of eggs with chromosome fragments dropped and did not occur at all in eggs fertilized with sperm treated 3.5 minutes. sperm irradiated 4.0.minutes However,.it appeared again in those fertilized with. The genetically inactivated sperm nucleus was visible in many eggs as a palestaining, 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 I (bivalents) ofthe female gamete to dissociate into Metaphase II-like clusters of 20 chromosomes (Table 3). 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 pseudo-metaphase 11 configuration~.fiveminute treatment times of 8,000, 12,000 and 14,000 PSI caused most eggs to advance to the Metaphase II-like configuration without any fertilization. fully consistent over trials at different PSI-time combinations. Results were not High,pressure treatment for more than 5 minutes,. however, seems only to scatter intact bivalents ' about the egg. Itis not known what, if any, portion of such eggs can complete - 4 -

6 ( meiosis andclecive. " 4It When early cleavage eggsare subjected to high PSI, the effect is to produce a colchicine-like metaphase arrest of the chromosomes (Table 4). At extremely high PSI there was a slight amount of chromosome breakage, pulverization, or scattering. Treatment of eggs at later cleavage stagesproduced the same results (Table 5). High pressure clearly has a strong effect on the spindle"in both the meiotic Metaphase I eggs, and in the 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 eggsbegan androgenic development. This i5 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 ch~omosomes. 'About 30% contained partially deteriorated chromosomes" of the female zygote. About 13% of the eggs began androgenic development, and"in 5% this was progressing normally towards cleavage (based on 128 eggs examined 6 hours post-fertilization). In these androgenetic eggs, the full complement of chromosomes of thefemale seemed,to'have beendestroyed. 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: As seen in Table 6, eggs irradiated for 5 minutes tend tobe polyspermie. Sometimes close to 100 sperm penetrated" these eggs. The male nuclei developing from such fertilizations were readily discernible in eggs fixed 1.5hours after fertilization. However, 7 of 30 eggs scored (23%) were penetrated by a single sperm, the developing - 5 -

7 "I nucleus of which was clearly visible. Two of these contained fragments of female chromosomes incompletely destroyed by prior irradiation of the egge :. -\...-. '.. By 6 hours after fertilization, the sperm in almost all such eggs had developed ".' elongated, pro-metaphase-like;chromosomes.,i.. chromosomes ~cattered Polyspermie eggs often had 100's of such about their cy~oplasm.even,so, 22% of the eggs.were observed to be clearly initiating androgenetic development. About half of these e1ther were I, '. in ana- or telophase or had metaphase chromosomes doubled for a mitotic division., ". of 6 cleavages observed were quite normal. Discussion This first,thorough ~ytological examinatio~ to be conducted on ~ny eggs of an aquaculture species in the.process ofgynogenetic stimulation wi~h Two irradiated sperm, raises the question whether thetotal. destruction of the male complement of sperm is. ~'.. '.. possible.. at levels.. ofirradiation., which still leave.' the' sperm capable of penetrating.\. ". and stimulating the female gamete to develop. Any inferior ~erformance of gynogenetic fish may be influenced then by fragment chromosomes from the male, or less likely,.. I _, there may be a positiveinfluence,of persisting fragment chromosomes ~n,viability., This study also shows that there is a high level of mitotic instability 11,'.' t" intrinsically associated with;the haploid state. This could be due to altered ratios I of chromosome. number to spindle masse.' '" Androgenesisis stimulated, in.the oyster less frequen~ly than gynogenesis.. ~. 'ț still occurs though, at level~. 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 :. '..,." i an almostexactreplica of.the mother (clones). This deserves further study. Natural. ~ t. I, parthenogenesis occurs in some invertebrate.groups,through suppression of Metaphase.;,. I ' I. Possibly though oyster eggs, subjected to high pressures and the likely attendant, ', delays in development would not remain viable.,,, ~ It - 6 -

8 4It Ultravidlet. light seems to be more effective in ~estruction of"the ge~etic, material of sperm without inactivating the sperm physiologically than does X-irradiation earlier used in a study on the oyster (Stiles, 1978). 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. In 17 different mass spawnings of a total of 835 wild Long Island" Sound oysters, a combined total of 6% of 1624 early cleavage eggs examined chromosomally were spontaneous haploids (Stiles and Longwell, 1973; Longwell, C.M. 1983jF:9). polyploidy occurred at a much lower frequency, about 1.5%. with a few extra or missing chromosomes) were about 3% and mosaics, about 1.5%. mass spawning to mass spawning. Aneuploid eggs (those Spontaneous of all examinedcytologically, This naturally occurring heteroploidy varied widely from In full-sib crosses of the American oyster, the spontaneous incidence of parthenogenesis appeared to be increased greatly (Longwell and Stiles, 1973). The experimental observations reported here, and also these instances of spontaneous parthenogenesis in American oyster eggs, both suggest that shellfish as well as finfish afford opportunities for chromosome engineering. Opportunities, of course, depend further on the ability of such eggs to develop and to do so be fore the egg ages too much,andjor upon the ability of the oyster to tolerate genetic homozygosity. 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 pe1ecypod mo11usks, a1though a study by Stanley et~. that oysters can tolerate induced polyploidy. (1961) shows The greater fecundity of pelecypod - 7 -

9 mollusks would afford some opportunities for recovery of such embryos even if their viability were much lower than in fish. Frequencies of spontaneous haploidy in oyster eggs suggest further that it would be worthwhile to examine young undersized spat, normally overgrown by other spat in regular culture practice,toascertain if these are haploids

10 , Table 1. Cytological examination of fertilization of American oyster eggs.withuv-treated sperm Minutes of irradiation Fertilization Fertil ization, and activation no activation No~ eggs % No. eggs % Polyspermy No.fertilization No. eggs % No. eggs %

11 Tab1e 2. Cyto1ogica1 examination of gynogenetic stimulation of American oyster eggs with UV-treated sperm No. two polar No. normal No. hap10ids No. normal No. dip10ids No. hap10ids with some Treat- No. eggs No. hap1o- dip10ids body dip10ids gynogenetic hap10ids with chroma tin irregu1ar ment examined dip10 with two with chromowith one. gynogenetic fragments of distribution time mosaics polar bodies some abnorma1- with two ities polar body polar bodies bridges of chromosomes '

12 r Table 3. Meiotic stimulatory effects of high pressure on unfertilized Metaphase I bivalents of the American oyster PSI 6,000 8,000 About 80% eggs in Meta 11 9,000 10,000* 12,000* About 80% eggs in Meta 11 14,000* Almost all eggs in Meta 11 5 Minutes 10 Some bivalents dissociated Some bivalents dissociated All eggs have scattered bivalents 20 Intact bivalents scattered Intact bivalents scattered * Same small incidence of a physiological-like effect on chromosomes - negligible

13 Table 4. Spindle disruptive effect of high pressure on early cleavage eggs'of the American oyster PSI.for 5 min Control 2,000 3,000 6,000 8,000 10,000* 12,000* 14,000* Effects on mitotic apparatus Normal array of normal mitosis Slight to no discernible effect Slight to no discernible effect All divisions in colchicine-like metaphase arrest All divisions in colchicine-like metaphase arrest All divisions in colchicine-like metaphase arrest All divisions in colchicine-like metaphase arrest All divisions in colchicine-likemetaphas~ arrest *Some evidence for chromosome breakage, pulverization and scattering negligible Table 5. Spindle disruptive effect of high pressure on mid-cleavage eggs of the American oyster PSI for 7 min Control 11,000* 12,000* 13,000* Effects on.mitotic apparatus Normal array of normal mitoses Most divisions in colchicine-like metaphase arrest All divisions in colchicine-like metaphase arrest All divisions in colchicine-like metaphase.arrest.. * Some evidence for some irregular prior mitoses

14 , Table 6. Androgenetic stimulation of oyster eggs after their ultraviolet treatment for 5 minutes 1.5 hours post-fertilization with untreated sperm Total 30 eggs scored 1 - unfertilized 22 - polysp~rmic 2-1 normal sperm nucleus with potentially viable fragments of the female's chromosomes 5-1 normal sperm nucleus 6 hours post-fertilization Total 64 eggs scored 1 - undeveloped sperm nucleus 49 - numerous chromosomes developed from polyspermie fertilization 1-10 chromosomes from single male nucleus plus 1 deteriorating male nucleus 5-10 chromosomes from single. male nucleus 2-10 chromosomes from single male nucleus doubled for cleavage division 4 - grossly abnormal cleavage 1 - normal cleavage with 20 chromosomes 1 - normal cleavage with 10 chromosomes Control - Normal development at normal rate 1.5 hours - early cleavages 6 hours - about 64-cell-stage embryos

15 REFERENCES. Kirpichnikov, V.S Genetic bases of fish selection. Translated from the Russian by G.G. Gause. 'Springer-Verlag, New York. 410 pp. - Longwell, A.C. and 5.5. Stiles. 1968a. Fertilization and completion of meiosis in spawned eggs of the American oyster, Crassostrea virginica Gmelin. Caryologia 21: Longwell, A.C.and 5.5. Stiles. 1968b. Removal of yolk from oyster eggs by Soxhlet extraction for clear chromosome preparations. Stain Technology 43: Longwell, A.C. and 5.5. Stiles Gamete cross incompatibility and inbreeding in the commercial American oyster, Crassostrea virginica Gmelin. Cytologia 38: Longwell, A.C., 5.5. Stiles and D.G. Smith Chromosome complement of the American oyster Crassostrea virginica, as seen in meiotic and cleaving eggs. Can. J. Genetics and Cytology 9: Purdom, C.E Genetic engineering by the manipulation of chromosomes. Aquaculture 33: '. Stanley,J.G.~;S.K. Allen, Jr. and H. Hidu Polyploidy induced'in the American oyster, Crassostrea virginica, with cytochalasin B. Aquaculture 23: " Stanley, J.G. and K.E. Sneed Artifical gynogenesis and its application in genetics and selective breeding of fishes. In: J.H.S~ Blaxter (Editor), The Early Life History of Fish. Springer-Verlag--,Berlin, pp '.. ~ ', ; Stiles, s ' Conventional and experimental approaches to hybridization and inbreeding research inthe oyster.]n: J.W. Avault (Editor), Proc. Ninth Annual Meeting, World Mariculture Society, pp Stiles, 5.5. and A.C. Longwel1, Fertilization, meiosis and cleavage in eggs from large mass spawnings of Crassostrea virginicagmelin, the commercial American oyster. Caryologia 26: ,'