Copyight 0 1994 by the Society of America

Sex Determination and Polyploid Gigantism in the Dwarf Surfclam (Mulinia lateralis Say)

Ximing Guo and Standish K. Allen, Jr. Haskin Shellfish Research Laboratory, Rutgers University, Port Nom's, New Jersey 08349 Manuscript received February 1, 1994 Accepted for publication August 20, 1994

ABSTRACT Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogeneticand triploid M. lateralis were successfullyinduced. For gynogenesis, eggs were fertilizedwith sperm irradiated with ultraviolet light and subsequently treated with cytochalasinB to block the release of the second (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogeneticdiploids were female, and there was no significant difference (P> 0.05) in sex ratio between diploids and triploids. These results suggestedthat the dwarf surfclam may havean XX-female, XY-male sexdetermination with Ydomination. Compared with diploids, triploids had a relative fecundityof 59% for females and 80% for males. Eggsproduced by triploid females were53% larger (PC0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P> 0.33) different from normal diploid females, suggestingthat was minimal in I1 gynogens. Triploid clams were significantly larger (P< 0.001) than normal diploids. We hypothesize that the in- creased body-size in triploids was caused by a polyploid gigantism due to the increased volume and a lack of cell-number compensation.

VER the last decade, set manipula- et al. 1993) and the Pacificoyster (Guoand ALLEN 0 tion in mollusks has received muchattention. 1994b). Those developments suggest that techniquesof Most ofthe researchhas focused on thedevelopment of chromosome set manipulation have reached a level of techniques for producing triploids and gynogens, pri- maturity in mollusks. However,the performanceof trip marily intended for potential applications in aquacul- loid, tetraploid and gynogenetic diploid mollusks has ture. Triploids have been successful in the culture of not been well documented. The growth of triploids is such as the Pacific Crassostrea gigas, for relatively wellstudied and has been reported in several which sexual maturation of diploids is a problem for species (BEAUMONTand FAIRBROTHER1991). Studies on marketing (ALLEN et al. 1989). Gynogens have potential the sterility of triploids have been limited primarily to for the rapid productionof inbred lines (PURDOM1983). histological examination of gonads. Fecundity and re- Chromosome set manipulation is also an important productive potential are estimated in only one species, tool for genetic analysis. The production of polyploids the Pacific oyster (Guo andALLEN 1994a). Observations has been useful in studies on effects ofchanges in chro- on the performance of tetraploid and gynogenetic mol- mosome number on development (FANKHALJSER1945). lusks are very preliminary (FUJINOet al. 1990; Guo and There have been attempts to evaluate the evolutionary GAFFNEY 1993;SCARPA et al. 1993; Guo andALLEN 1994b). significance of in , but they are lim- A majordifficulty in evaluating the performance of ited by our poor understanding of the biology and re- polyploid and gynogenetic mollusks is their long genera- productive genetics of polyploid animals (SCHULTZ1980; tion time, usually 1-2 years. To overcome this constraint, BOGART1980; Guo and ALLEN 1994a).The production of we used the dwarf surfclam (Mulinia lateralis Say) as a gynogens can be used for gene mapping and to study model species to study effects ofvarious chromosome set genetic sex determination which is still largelyunknown manipulations. M. lateralis has a short generation time of in mollusks (HALEY 1977; 1979; ALLEN et al. 1986). about 3 months and is easily cultured in the laboratory In mollusks, triploidy has been induced inover a (CALABRESE 1969). In this study, M. lateralis was used to dozen of species (see reviewby BEAUMONTand FAIR- document performance of gynogens and triploids. BROTHER 1991). Successful gynogenesis were also re- ported in the Pacific abalone (FUJINOet aZ. 1990) and the MATERIALS AND METHODS Pacificoyster (GLJOet al. 1993). Recently,viable tet- M. lateralis parents used in this study werethe F,s from a raploids were also reported in the blue mussel (SCARPA random mating of animals obtained from the state ofVirginia.

Genetics 138: 1199-1206 (December, 1994) 1200 X. Guo and S. K. Allen

Gametes were obtained by dissecting gonads, and thesomatic tissuewas saved for allozymeanalysis. Eggs were passed through a 85-pm Nytex screen to remove large tissue debris, and rinsed on a 25-pm screen. Sperm suspensions were passed through a 15ym screen to remove large debris. Seawater used for rearing larvae was filtered to 2 pm, and the salinity was about 25 ppt. Fertilization and treatments were conducted at 24-26". Gynogenesis was induced by fertilizing eggs with sperm ir- radiated by ultraviolet (W)light. Two &watt shortwave UV tubes (G8T5, VWR) were used in the irradiation chamber. To determine the appropriateUV dosage, sperm were exposed to W light for 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0 min at an intensity of 1300-1500 pW/cm2. The UV intensity was meas- ured by a Black-Ray UV meter 0225, UW,Inc.) and adjusted by varying the distance between sperm and UV light tube. For the dosage experiment, eggs from four females were pooled and then divided equallyinto nine groups, and sperm from six I maleswere combined. The sperm density was adjusted to D about 100 million/ml at a standarddepth of 1 mm (Guo et al. UV Duration (min) 1993). The success of gynogenesiswas judged by fertilization FICUKE1.-Fertilization, survival and induction of haploids success and development of haploids. Fertilization success was determined at 90-120 min post-fertilization (PF) as the pro- with sperm of M. lateralis exposed to different durations of UV light (1400 pW/cm2). portion of eggs undergoing polar body release or cleavage. The induction of haploid development was determined by of seawater and counted in two subsamples of 0.5 ml. For chromosome counts of 20 2-4cell embryos per group (Guo males, sperm were suspended in 1 liter of seawater, and four and ALLEN 1994a). The optimum UV dosage was used to pro- to five subsamples were counted using a hemacytometer. duce gynogenetic diploids in subsequent experiments. Gynogenetic diploids were induced by blocking polar body I1 (PB2) with cytochalasin B (CB) in eggs fertilized with UV- RESULTS irradiated sperm (THORGAARD1983; GUOet al. 1993). Triploids W dosage: Fertilization success decreased gradually were induced by blocking PB2 with CB in eggs fertilized with with increased W dosage (Figure 1), decreasing to 49% untreated sperm (ALLEN et al. 1989). To block the release of PB2, fertilized or activated eggs weretreated with 0.5mg/liter when sperm were irradiated for 3 min. With 4 min of W CB dissolved in 0.5 ml dimethyl sulfate (DMSO), for 15 min irradiation, only 2% of the eggs developed, and expo- beginning at 30 min PF. Four experimental groups were pro- sures of 5 min or longer completely destroyed the duced with each pair of parents (replicate): In, a haploid sperm's ability to activate eggs. group in which eggs werefertilized with UV-irradiated sperm Chromosome numbers in the resulting embryos in- and allowed to develop without CB treatment; 2n, a normal fertilization used as a diploid control; 2ng, a gynogenetic dip- dicated that all W dosages tested in this study were ef- loid group; and 313, a triploid group. The four groups were fective in inactivating sperm (Figure 1). replicated five times using different pairs of parents. Even when sperm were irradiated for as short as 30 sec, In each replicate, chromosome number of 100 embryos no diploid development occurred. The dwarf surfclam (2-4 cells) in thehaploid groups was determined to verify the has a diploid chromosome number of38 (In = 19) inactivation of sperm chromosomes. Embryos werecultured in (WADAet al. 1990). Out of 20 embryos analyzed from the 15-liter buckets at a density of 50-67 embryo/ml; approxi- mately equal density was maintained in allfour groups. 30-sec irradiation, 19 had exactly 19 chromosomes, and of 24hr-old larvae was analyzed by flow cytometty and survival one had 20 chromosomes. Among all embryos (100) of fertilized eggs to D stage (24hr PF), day 3 and day 8 was analyzed from five treated groups, 97 had 19 chromo- determined for all groups. For counting, larvae werecollected somes, one had 18 chromosomes and two had 20 chro- on a Nytex screen of proper size and rinsed to a known volume mosomes. Examples of haploid and diploid metaphases of seawater (usually 1-10 liters). Three subsamples of 1 ml were placed in countingchambers and counted under micro- are presented in Figure 2. The UV-dosage experiment scope. The average of the three subsamples was used to de- was not repeated because ofthe consistency of the initial termine the total number of larvae in that group. After meta- experiment. A dose of 1.5-min W irradiation (1400 morphosis (8-10days PF), clamswere transferred to pW/cm2) to sperm (108/ml, 1-mm thick) was consid- recirculating systems with sand substrate where all groups ered optimum for inactivation and used for subsequent within each replicate shared the same feeding reservoir, and approximately the same amount (liters) of were deliv- experiments. In the untreated control,no haploids were ered to each reservoir. found, and 95% of the embryos had exactly 38 chro- At 3 months PF, clams in all groups were sampled. The mosomes. One embryo in the control group had 39 ploidy of sampled clams were determined by flow cytometry chromosomes. (ALLEN 1983; Guo et al. 1993). Length and whole body weight As expected from the chromosome data,survival to D (wet) were measured. Sex was determined by microscopic ex- stage dropped sharply to zero at Wa duration as low as amination of gonads. Fecundity of a subsample of clams was also determined by dissecting gonads and enumerating ga- 0.5 min (Figure 1), suggesting haploids are incapable of metes obtained. For females, eggs were suspended in 750 ml reaching D stage. With high dosages used in this study, G ynogenetic and Triploid and Gynogenetic Mulinia 1201

TABLE 1

Number of eggs used, percent fertilizationand cumulative survival of fertilized to D stage, day 3 and day 8 in experimental groups by replicates

Group" Eggs (10') Fertilization D stage Day 3 Day 8

In 1 I 70 97 0 2 69 100 0 3 98 95 0 4 30 9.5 0 5 63 9 1 0 Mean 0 2n 1 170 100 65.270.0 13.0 69 100 2 69 68.168.1 94 98 3 94 66.9 63.9 30.9 4 30 100 40.440.4 38.6 5 63 98 58.66.5.5 62.2 59.2 27.5 Mean 59.2 62.2 2ng 1 0.3 567 1.9 91 0.3 2 41 I 100 5.0 0.8 3 983 96 7.01.5 2.0 4 301 98 4.5 1 .0 0.4 5 634 9 3 0.5 0.04 Mean 3.8 0.8 0.7 3n 1 283 100 3.6 2.0 2.0 2 137 100 11.4 11.4 295 96 3 295 40.044.3 31.1 4 60 98 29.3 12.428.5 127 98 5 127 8.8 11.7 Mean 20.1 18.1 15.2 FI(:I.KI.2.-Mct;~ph;tscs lrom (X) haploid 2-4 CCII crllbryos of Mulinin l(l/uu/i.s procIucecl bv W-irradiated sperm (In = ' In = gynogenetic haploid; 2n = diploid control; 2ng = gynoge- 19), and from (R) gynogenetic diploid produced from reten- netic diploid; and 3n = triploid. tion of the second polar body after CB treatment (2n = 38). the average, 27.5% for the diploid control,0.7% for the gynogenetic groups, and 15.2% for the triploid groups. we saw no evidence for spontaneousincrease in survival Replicates 2 and 5 were discontinued at day 3 due to known as the Hertwig effect. low survival, lack of chromosome counts of haploids, Inductionand survival of gynogensand triploids: and space limitations. Larvae in the remaining three The UV treatment used in the production of gynoge- replicates (2n, 2ng and 3n groups per replicate) meta- netic diploids effectively inactivated spermchromo- morphosed at day 8-10 PF and were reared to sexual somes. Chromosome numberswere determined forem- maturation. After metamorphosis, young clams were bryos in three of the five haploid groups (replicates 1, transferred to a recirculating system for rearing. There 3 and 4), 100 per group.The 300 embryos analyzed con- was virtually no post-larval mortality. sisted of 281 (93.7%) haploids, 18 (6.0%) aneuploids At day 30 PF,the ploidy of 25 clams were determined and 1 (0.3%)triploid. All aneuploids had low chromo- in each group by flowcytometry. Clams from the diploid some numbers close to haploidy (In = 19): 5 (1.7%) control and gynogenetic groups were all diploid. In trip- with 18 chromosomes, 10 (3.3%) with 20 and 2 (0.7%) loid groups, diploidsand triploids were the only types of with 21. One aneuploid (0.3%)was mosaic with 18 and ploidy identified. The percentages of triploids were 88, 20 chromosomes. 33 and 100% for the threereplicates (Table 2). Approxi- The ability of the sperm to fertilize eggs was unaf- mately the same percentages of triploids were observed fected by UV treatment, and fertilization in all groups at 3 months PF in all three replicates. was high, ranging from 91 to 100% (Table 1). Survival Sex ratio, fecundity and body size: were ob- of fertilized eggs was variable among In, 2n, 2ng and 3n served in the majority of clams in all groups at three groups. In the normal diploid control(2n), theaverage months PF. The percentage of immature clams was ap survival to D stage was 62.2%. Haploid groups (In) had proximately the same in all experimental groups (Table no survival to D stage in all five replicates. On the other 3). Diploid clams were 40% female, 50% male and 10% hand, blocking PB2 in the gynogenetically activated eggs immature. If we could assume the immature clams were (2ng) allowed 3.8% of them to survive to D stage, rang- females, the diploids would have an exact 1:l sex ratio. ing from 0.5 to 7%. In triploid groups (3n), an average In the gynogenetic diploid groups,however, not a single of 20.1 % of the fertilized eggs developed into D stage male was found among 225 clams examined, and all (Table 1 ) . The same pattern of survival persisted to day clams whose sexcould be determinedwere females. Im- 3 and day 8 PF. At day 8 PF, cumulative survival was,on mature clams accounted for16% of gynogens. Although 1202 X. Guo and S. K. Allen

TABLE 2 TABLE 4 Percent triploids inducedin three replicates determined by flow Fecundity of normal diploid (2n),gynogenetic diploid (2ng) and cytometry at one and three months post-fertilization (PF) triploid (34 M. lateralis at three month of age

1 Month PF 3 Months PF Group n Mean" CV% Significance Replicate n % n % 2n Female 297.4 25 55 b 27 235.42ng 27 82 ab 1 25 88 75 92 24 3n 174.3 70 a 3 15 33 75 21 Male 2n2.487 25 51 100 75 100 4 75 25 100 3n 1023 2.001

a Means for females = XlO', and means for males = X109. TABLE 3 Letters designate significant differences in means among females, Sex-ratio of normal diploid (2n), gynogenetic diploid (2ng), and as indicated by the Tukey HSD multiple comparison test at a confi- triploid (3n) M. lateralis at three month of age dence level of 95%. The difference between male 2n and 3n was not significant (P> 0.05). Group Female (%) Male (%) No gametes (%) Total possibility that they might be genetic heterogeneousand 2n 89 (40) 113 (50) 23 (10) 225 different from normal diploid controls (due to gyno- 2ng 188 (84) 0 (0.0) 37 (16) 225 3n 46 3n (29) 93 (58) 21 (13) 160 genesis, for example). Effects of gynogenesis and trip loidization were tested in two separate analyses (Table 5). For analysis on gynogenesis, only gynogenetic and the difference in sex ratio between diploids and triploids normal diploid females were used. In both length and was not statistically significant (P= 0.09), triploids did weight measurements, analysis of variance (ANOVA) show a significant (P< 0.05) deviation from the 1:l sex showed that gynogenesis had no significant effects (P> ratio favoring more males, even assuming all immature 0.80). Effects due to replicate (or family) were signifi- clams were females. No hermaphrodites were observed cant for length (P = 0.001), but not for weight (P = in any group in this study. 0.184) (Table 5). Variation among replicates might be On average, normal diploid M. lateralis produced caused by genetic differences among females or random 3 X lo5 eggs or 2.5 X lo9 sperm per individual (Table environmental factors during replication. The interac- 4). Comparatively, gynogenetic diploids produced fewer tion between gynogenesis and replicate was also signifi- eggs (2.4 X lo5), which corresponds to a relative fecun- cant for both measurements (P< 0.01). In fact, when dity of 79%. The difference in fecundity between gyno- three replicates were examined individually with a two genetic and normal diploids was not statistically signifi- sample t-test (at 95% confidence level), gynogens were cant however (P = 0.36). On the other hand, triploid significant smaller than normal diploids in the first rep- females produced significantly (P = 0.03) fewer eggs licate (11.9 mm and 315 mg us. 13.1 mm and 410 mg), (1.7 X lo5), which correspond to a relative fecundity of significantly larger than normal diploids in the second 59%. Triploid males produced fewer sperm with a rela- replicate (14.1 mm and 434 mg us. 13.2 mm and 359 tive fecundity of 80% (Table 4), but the differences mg), and not significantly different from normal dip- between triploid and diploid males was not statistically loids in the thirdreplicate (13.0 mm and 367 mgvs. 13.0 significant (P = 0.10). When fecundity was analyzed mm and 371 mg). Apparently, this interaction was due per unit body mass, however, both triploid males and to variation in gynogens, not normal diploid females. females had significantly (P = 0.000 and P = 0.001) Overall, gynogenetic females measured 12.9 mm and reduced fecundity than normal diploids. 367 mg, which were not significantly different from the Diameters of ten eggs were measured for 36 normal 13.1 mm and 372 mg of normal diploid females when diploid, 17 gynogenetic diploid, and 24 triploid females. tested by a two sample t-test (P= 0.332 for length, P = Eggs from normal diploids averaged 49.1 2 3.1 (SD) pm, 0.794 for weight) (Table 6). Interestingly, gynogens (in- and eggs from gynogenetic females were approximately cluding immatures)were significantly(P< 0.01) smaller the same size, 48.6 ? 3.1 pm. Eggs from triploids which than normal diploids of both sexes (including imma- measured 56.6 5 3.5 pm in diameter were significantly tures), suggesting males were larger than females in nor- (P< 0.000) larger than those of normal diploids. Com- mal diploids. pared with normal eggs, eggsfrom triploids were 15.3% For analysis on triploidization, all individuals from larger in diameter,an estimated 53% increase in diploid and triploid groups were used (including im- volume. matureclams). In both length and weight measure- At 3 months PF, length and weight measurements of ments, the ANOVA showed that effects due to ploidy 75 clams weretaken for each group.Clams from triploid (diploid or triploid) were significant (P = 0.012 for groups were individually confirmed by flow cytometry. length; P = 0.005 for weight). Effects due to replicate were Diploids from triploid groups (6, 59 and 0 for three significant for weight (P= 0.01), but notfor length (P= replicates, respectively) were excluded because of the 0.085) (Table 5). There was no significant interaction Gynogenetic and Triploid Mulinia 1203

TABLE 5 Analysis of variance for length and weight of three families of gynogenetic (Zng), diploid (2n) and triploid (3n) M. lateralis groups

Length (mm) Weight (mg)

Source d.f. MS F ratio P MS F ratio P

Gynogenesis: Gyno (G,fixed) 162 10.825.o 0.1 0.0 0.980 Replicate (R,random) 0.184 25.9 1.7 2 32,708 0.001 7.2 5.5 0.004 127,238 6.6 0.002 GXR6.6 19.7 127,238 2 0.004 5.5 271 3.6 19,215 3.6 Error 271 Triploidization: 524.6 70.9 0.012 4,729,588 307.1 0.005Ploidy 307.1 (P,fixed) 4,729,588 0.012 70.91 524.6 Replicate (R,random) 0.01015.5 4.6 2 234,837 0.085 2.5 2 7.4 1.2 0.309 15,400 0.3 0.738 0.3 15,400 0.309 1.2 PXR 7.4 2 Error 379 6.3 50,656 Effects of gynogenesis and triploidization were tested in two separate analyses. For gynogenesis, only females from gynogenetic and normal diploid groups were used. For triploidization, all individuals from triploid and normal diploid groups were used. TABLE 6 respectively (Table 6).The difference was highly signifi- Length and weight measurements of gynogenetic (2ng), normal cant (P= 0.000) as indicated by a two-sample t-test.The diploid (2x1) and triploid (3n) M. lateralis Say at 3 months of age size difference between triploids and normal diploids were obvious without measurements, and in fact many Group n Mean CV (%) P valuea of the triploids were gigantic compared with any of our Length (mm) in-house diploid populations, domestic or wild. 2n female13.1 89 14.1 2ng female12.9 188 0.332 16.2 DISCUSSION 2n allb 13.3 225 15.9 3n all 16.2 160 18.5 0.000 Gynogenesis: The UV treatment (1400 pW/cm2, 1.5 min, sperm/ml, 1 mm deep) defined in this study Weight (mg) lo8 2n female37.6 372 89 was effective in inactivating chromosomes of M. lateralis 188 367 39.6 0.7942ng 39.6 female 367 188 sperm. In the haploid groups, none of the 300 early 2n all 225 390 42.6 390 225 all 2n embryos examined were diploid. Embryos with 20 chro- 3n all 160 672 43.6 672 160 all 3n 0.000 mosomes (n + 1) may or may not have inherited the The P value is from a two-sample t-test. extra chromosome from sperm.Such a small frequency bFor 2n and 3n, all female, male and immature clams were in- cluded. (3%)of chromosome additioncould be caused by spon- taneousnondisjunction of maternal chromosomes between ploidy and replicate in both length (P= 0.31) (HECHTand HECHT1987). No chromosome fragments and weight (P = 0.74) measurements, suggesting trip were observed as suggested by previous studies (XUet al. loids were always different from diploids regardless of 1990; Guo et al. 1993). replicates (or families). Sex was not included as a factor Blocking PB2 restored diploidy in the gynogenetically in the ANOVA because of the presence of immature activated eggs and also increased their survival. Allozyme clams and their daerential distribution among three inheritance in gynogens is being examined, and for 12 replicates. The immature clams were small individuals polymorphic loci examined so far, all paternal alleles that had not reached sexual maturation, and their ex- were absent in all gynogens from all three replicates, clusion would have created bias in the ANOVA.Al- suggesting that thegynogenesis obtained in this study is though sex could not be includedin the ANOVA, it was very “clean” (X. GUOand S. K. ALLEN, manuscript in apparent that thetwo sexes were different in body size. preparation). Thesuccess ofgynogenesis was suspected For all normal diploids whose sex was identified, males even before the electrophoreticconfirmation. First, sur- averaged at 14.0 mm and 439 mg, females averaged 13.1 vivors (diploid) were found only in CB-treated groups, mm and 371 mg, and the difference was significant not in haploid controls. Second, 2ng survivors were all (P<0.01) when tested by a two sample t-test. In triploids, female, contrasting to the normalsex ratio in the control males were also larger than females, and the difference groups. In such as salmonids and tilapia, gynoge- was significant in weight (P= 0.02), but not in length netic diploids were also all females (CHOURROUTand (P= 0.10). QUILLET1982; MAIR et UI!. 1991) Triploids were significantly (P< 0.001) larger than Compared with normal diploids and triploids, the sur- diploids in either sexes. Overall (including both sexes vival of gynogenetic diploids was low (0.7%). Reduced and immature ones), triploids averaged at 16.2 mmand survival is common for gynogenetic fish and mollusks, 672 mg, which were 22 and 72% larger than normal and itis commonly believed to becaused by inbreeding diploids (13.3 mm and 390 mg) in length and weight, depression (SCARPA1985; MA 1987; Xu et al. 1990; FUJINO 1204 X. Guo and S. K. Allen

et al. 1990). On the other hand, there were several in- loids, which agrees with a previous study in the same dications thatinbreeding depression is not a species (RUPRIGHT1983). Besides M. lateralis, triploid cause for the reduced survival. First, gynogenesis and mollusks were significantly larger than diploids in al- androgenesis using inbred strains did not lead to sig- most all species studied, including theAmerican oyster nificant improvement on survival (SCHEEIZERet al. 1986; (Crassostrea virginica)(STANLEYet al. 1984),bay scallop GUOet al. 1993). Secondly, because of high recombi- (Argopecten irradians) (TABARINI1984), thePacific oys- nation frequencies, thelevel of inbreeding in meiosis I1 ter ( Crassostrea gigas) (ALLEN and DOWNING1986), the gynogens was low (fixation index = 0.26), similar to that scallop (Chlamys nobilis) (KOMARUand WADA1989), of a sister-brother mating (Guo and GAFFNEY1993). Al- the pearl oyster (Pinctada martensii)UIANC et al. 1991) ternatively, Guo et al. (1993) suggested that damaged and the scallop ( Chlamysfarreri) and the oyster (Cras- DNA fragments from sperm irradiationcan potentially sostrea rzuularis) (R. C. WANC,personal communica- be a cause for the reduced survival. tion). Triploids were not significantly different from Overall, there was no significant difference in growth their diploid controls in the soft-shell clam (Mya are- measurements between gynogens and normal diploid naria) (MASON et al. 1988), andin one of the studies of females, suggesting again that inbreeding depression is American oyster (STANLEYet al. 1981). In thehard-shell minimal for meiosis I1 gynogens. On the other hand, the clam (Mercenaria mercenaria) , triploids were smaller significant interaction between gynogenesis and repli- than their diploid controls (HIDUet al. 1988). cates (or females) indicated that the performance of Two hypotheses have been proposed to explain in- gynogens might be a functionof the maternal . creased body size in triploid mollusks. The first is the In the Pacific oyster, gynogenetic diploids were either heterozygosity hypothesis, proposed by STANLEYet al. smaller than or not differentfrom diploid controls (Guo (1984), stating that the increased body size in meiosisI and GAFFNEY1993). triploids (produced by blocking PB1) was caused by in- Sex determination: The fact that gynogenetic dip- creased heterozygosity. This hypothesis may also explain loids were all female strongly suggests that, in M. late- differences between diploids and meiosis I1 triploids be- ralis, females are homogametic (XX). Also the fact that cause theoretically, all triploids (either from meiosis I or no intersex individuals were found amongtriploids sug- meiosis I1 inhibition) are more heterozygous than dip- gests that themaleness-gene is dominant, so that XXXis loids (ALLENDORF and LEARY1984). Supporting the het- female, and XXY is male. Therefore, we propose thatsex erozygosity hypothesis, meiosis I triploids were usually in M. lateralis is determined by a XX-female, XY-male larger than meiosis I1 triploids andnormal diploids mechanism with a dominant maleness gene(s), similar (STANLEYet al. 1984; JIANG et al. 1991; BEAUMONTand to that in and othervertebrates. Under such KELLY 1989). The second hypothesis, originating from a system,sex determination in triploids induced by finfish work (PURDOM 1972),argues that the increased blocking PB2 should be unaffected. The slightly biased body size in triploids is caused by energy reallocation sex ratio of triploids observed in this study could be from gametogenesis to growth due to the sterility of trip- caused by differential mortality of triploid males and loids. Supporting the energy reallocation hypothesis, females, or sampling errors. Previous studies on the triploid Pacific were larger than diploids only of M. lateralis, however, found no evidence after spawning (ALLEN and DOWNING1986). In other for dimorphic sex chromosomes (MENZEL1968; WADA studies, energy reallocation was suggested because trip- et al. 1990). Sex determination in mollusks is largely loids grew faster than diploids around thetime of sexual unknown. In theAmerican oyster, it has been proposed maturation (TABARINI 1984;JIANC et al. 1991). that sex is determined by multiple loci (HALEY1977, We agree that both heterozygosity and energy real- 1979). In the soft-shell clam, an Xautosome determi- location may contribute positively to the overall per- nation, similar to that in Drosophila, has been proposed formance of triploids. However, we hypothesize that based on the presence of possible intersexes and ab- they are not the primary causes for the increased body sence of males among triploids (ALLEN et al. 1986). The size in triploids. First, the heterozygosity hypothesis was diverse sex determination among mollusks, if true, may proposed under the assumption that meiosis I triploids limit the generality of M. lateralis as a model species. are moreheterozygous than meiosis I1 triploids (STANLEY It is clear that there were differences in growth be- et al. 1984). This assumption is true only when the re- tween two sexes in M. lateralis, although incomplete combinant frequency is lower than 0.67 (GUO et al. maturation encounteredin this study prevented a more 1992). A preliminary study in the Pacific oyster esti- definitive analysis. If males are larger than females in mated that theaverage recombinant frequency ( r) over commercial species (such as hard clam and Manila seven loci was 0.74 (Guo andGAFFNEY 1993), suggesting clam), the creationof all male populations through an- that the heterozygosity of meiosis I triploids (1 - r/2) drogenetic YY-males should be useful. was lower than that of meiosis I1 triploids (r).Other Polyploid gigantism: Triploid M. lateralis produced studies found a lack of correlation between heterozy- in this study were significantly larger than normal dip- gosity and growth rate in meiosisI and I1 triploids UIANG and Triploid Mulinia TriploidGynogenetic and 1205

et al. 1991; MASON et al. 1988). Within natural diploid tion). In ourexperience with Pacific oysters,the adduc- populations, the correlationbetween heterozygosity and tor muscles of triploids are visibly larger than those of growth rate is weak, although generally (but notalways) diploids, and the adductor muscle of tetraploids were positive (GAFFNEY1990). It is hard to imagine that in- extraordinarily (or abnormally) large (GUOand ALLEN creased heterozygosity can increase the body size oftrip 1994b and unpublished).Interestingly, since the adduc- loids by 72% in M. lateralis (this study), 27-58% in tor muscle is the site of at least some glycogen storage Pinctadamartensii UIANG et al. 1991), 36% in Arg- in bivalves, its increased size has also been seen as an opecten irradians (TABARINI1984), 32-59% in Chlamys indicator of energy reallocation (TABARINI1984). nobilis (KOMARUand WADA1989), and 81% in Chlamys The expression of polyploid gigantism can be affected farreri (R.C. WANG,personal communication). Second, by environmental factors, which may explain the failure the energy reallocation hypothesis can not explain find- to recognize polyploid gigantism in previous studies in ings where triploids were significantly larger than dip mollusks. One of the factors, we speculate, may be nu- loids before sexual maturation UIANG et al. 1991; R. C. trition. Because triploid cells are larger, they may need WANG,personal commnication). Also in this study, trip more nutrients to grow and divide. In an environment loids were larger than diploids at 30 days PF,long before where food supplies are limiting, triploids may not be sexual maturation (data not presented here). Even dur- larger than diploids before sexual maturation. When ing maturation, it is difficult to understand how reallo- diploids attain sexual maturity, triploids may continue to cation of metabolic energy (from gonad to somatic tis- grow and surpass diploids, masking any affect of gigan- sue) could lead to a net increase in body size, unless tism. In more productive waters, triploid Pacific oysters diploids spawned and triploids did not. In this study, often grow faster than diploids even before diploids have neither diploids nor triploids had spawned at 3 months sexually matured (DAVIS1989; K. COOPER,personal of age (clams were reared inside the laboratory and communication). In this study, several factors may monitored daily). Clearly, the energy reallocation hy- have contributed to our finding of polyploid gigan- pothesis is not sufficient to account for the increased tism. First, food was not limiting. We fed experimental body size in triploids observed in this and otherstudies. clams daily from algal cultures (Isochrisis galbana and In view of the difficulty of the existing hypotheses to Chaetoceros calcitrans) maintained in our lab. Second, adequately account forincreased growth in triploid mol- neither diploid nor triploidclams had spawned prior lusks, we would argue that the increased body size in to sampling. triploid mollusks is caused by a polyploid gigantism, due Separation of the heterozygosity, energy reallocation to the increased cell volume and a lack of cell number and polyploid gigantism hypotheses is important for fu- compensation. Polyploid gigantism was probably dis- ture research and application of polyploidy in mollusks. missed earlyin mollusk workbecause of the assumption It is apparent that heterozygosity and energy realloca- that animals in general display a reduction in cell num- tion hypotheses are insufficient, and the polyploid gi- ber with increasing cell size. With our data and upon gantism hypothesis needs to beconfirmed by direct stud- further reflection, we think gigantism warrants another ies on cell size, cell number and organ size in diploids look. and triploids. Polyploid gigantism is a common feature in Fecundity of triploids: The relative fecundity of trip (BLAKESLEE1941; STEBBINS1956) and has also been ob- loids observed in this study is surprisingly high, 59% for served in some invertebrates such as the females and 79% for males. That fecundity in triploids (Artemia salina), the bagworm moth (Solenobia sp.), was so high and gigantism was still apparent strengthens the isopod Trichoniscus elisabethaeand Drosophila (re- our hypothesis over the energy reallocation hypothesis. viewed by Fankhauser 1945). In higher animals such as In the Pacific oyster, the relative fecundity of triploid fish and amphibians, however, the increased cell volume females was estimated to be only 2% (GUO andAILEN in polyploids is often compensated forby a reductionin 1994a). The production of large numbers of gametes by cell numbers so that the overall size of organs was un- triploids warrants further studies on their sterility.

changed (FANKHAUSER1945; BENFEYand SUTTERLIN We are grateful to AMY TAYLORfor assistance in larval and nursery 1984). We hypothesize that, in polyploid M. lateralis cultures and in the laboratory, and to ANN E. ARSENIU for general lab and possibly all mollusks, the cell number reduction is assistance. An anonymous reviewer offered constructive suggestions absent, producingpolyploid gigantism. Polyploid gigan- on data analysis. This is New Jersey Agricultural Station Publication NO. D-32100-2-94 and Contribution No. 94-04 of the Institute of tismmay not be apparent inall organs (FANKHAUSER Marine and Coastal Sciences, RutgersUniversity, supported by 1945). In bivalves, the adductor muscle seems particu- U.S. Department ofAgriculture/CSRS/OGPS 92-37203-7766 to S.RA. larly subject to polyploid gigantism. Adductor muscles of and X.G. triploids were 73% larger than those of diploids in the bay scallop (TABARINI1984), 96% larger in the scallop LITERATURE CITED (Chlamysfarreri)and 198% larger in the oyster (Crus- ALLEN, S. R, JR., 1983 Flowcytometry: assaying experimental sostrea rivularis) (R. C.WANC, personal communica- polyploid fish and shellfish. Aquaculture 33: 317-328. 1206 X. Guo and S. K. Allen

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