Tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis (Say 1822): effects of cytochalasin B duration

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1 doi: /j x Tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis (Say 1822): effects of cytochalasin B duration Huiping Yang & Ximing Guo Haskin Shell sh Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, Port Norris, NJ, USA Correspondence: X Guo, Haskin Shell sh Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University,6959 Miller Avenue, Port Norris, NJ 08349, USA. xguo@hsrl.rutgers.edu Abstract Di erent durations of a cytochalasin B (CB) treatment were tested for tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis Say. Cytochalasin B, 0.67 mg L 1, was applied to newly fertilized eggs at 8^10-min post fertilization and removed when in the untreated eggs: (1) Polar body1 (PB1) was released in 90% of the eggs and polar body 2 (PB2) began to form (T1); (2) Polar body 2 was released in about 25^30% of the eggs (T2); (3) Polar body 2 was released in about 70^75% of the eggs (T3); or (4) eggs began to enter mitosis I or the polar lobe began to form (T4). Three replicates were produced using di erent sets of parents. The ploidy of resultant larvae and juveniles was determined by ow cytometry. Blocking PB1 alone in T1 groups produced mostly tetraploids, and longer CB treatments in T2 and T3 resulted in increasing numbers of pentaploids. In T4 groups where both PB1 and PB2 were inhibited, larvae were predominantly pentaploids. Pentaploid larvae were arrested at the trochophore stage. The majority of tetraploid larvae died as trochophores, although a small fraction reached D-stage. Among 478 juvenile clams sampled from a T1 group, three (0.6%) were con rmed as tetraploids. This study shows that tetraploid embryos can be produced at high e ciencies (40^90%) by blocking meiosis I. Tetraploids produced by meiosis inhibition in normal eggs are viable in M. lateralis, but their survival beyond metamorphosis is extremely low. Keywords: tetraploidy, triploidy, cytochalasin B, meiosis, aquaculture, Mulinia lateralis Introduction Tetraploids are organisms with four sets of chromosomes, instead of the two sets found in normal diploids. Tetraploid shell sh are valuable, because they can mate with diploids and produce 100% triploids (with three sets of chromosomes) (Guo, DeBrosse & Allen 1996). Triploid shell sh are useful for aquaculture because of their sterility, superior growth, improved meat quality and sometimes increased disease resistance (Allen & Downing 1986; Guo & Allen 1994a; Hand, Nell, Smith & Maguire1998;Wang, Guo, Allen & Wang 2002). Despite intensive e orts, tetraploid induction remains a challenge in shell sh genetics and breeding. Only in three species of Crassostrea oysters, large numbers of tetraploids were obtained, and breeding populations were established (Guo & Allen 1994b; Eudeline, Allen & Guo 2000; Supan, Allen & Wilson 2000; Guo, Wang, Landau, Li, DeBrosse & Krista 2002; Allen, Erskine,Walker & Zebal 2003). Success in tetraploid induction in Crassostrea oysters was achieved using a novel approach ^ blocking polar body1 (PB1) in eggs from triploids fertilized with normal sperm (Guo & Allen 1994b). The Guo and Allen method may not work in species where triploid females produce no or few eggs. Methods for direct induction of tetraploids from normal diploids are needed for most molluscs. In marine bivalves, tetraploids can be produced by direct induction from diploids by meiosis inhibition, mitosis I inhibition, gynogenesis, or cell fusion (Beaumont & Fairbrother 1991; Guo 1991). While tetraploid embryos were readily produced, tetraploids failed to survive beyond larval stages in most species studied r 2004 Blackwell Publishing Ltd 1187

2 so far (Stanley, Allen & Hidu 1981; Arai, Naito & Fujino 1986; Yamamoto & Sugawara 1988; Stephens 1989; Guo, Hershberger, Cooper & Chew1994). A few viable tetraploids were produced by treatments aimed at blocking both polar body 1 (PB1) and 2 (PB2) in the blue mussel (Scarpa, Wada & Komaru 1993) and the dwarf surfclam (Peruzzi & Guo 2002). In the Manila clam, incidental tetraploids were observed among triploids produced by blocking PB2 (Allen, Shpigel, Utting & Spencer 1994). In the zhikong scallop, a small number of viable tetraploids were obtained by blocking PB1 (Yang, Zhang & Guo 2000b). Blocking PB1 and PB2 is expected to produce pentaploids (Cooper & Guo1989). The production of tetraploids by blocking PB1 and PB2 in two independent studies deserves further examination. Because meiosis in molluscs is not completely synchronized, it is possible that treatments intended for both PBs e ectively inhibit only PB1. It is also possible that blocking PB1 and PB2 is an e ective method for tetraploid induction, either because longer treatments a ect chromosome segregation producing true and viable tetraploids due to mechanisms not yet known. To determine if treatment length covering PB1and various proportions of PB2 release a ects the production of viable tetraploids, we tested four durations of a cytochalasin B (CB) treatment using the dwarf surfclam as a model species. Here we report the production of a few viable tetraploid clams from short (blocking PB1), but not from long treatments (blocking PB1and PB2). Materials and methods At 23ºC PB1 release PB2 release Mitosis I min T1 T2 T3 T4 Figure 1 Schematic presentation of meiotic events in Mulinia lateralis and treatment coverage for tetraploid induction by meiosis inhibition with cytochalasin B. Four durations of a CB treatment were tested. The four durations were designed to inhibit PB1 plus different proportion of PB2 (Fig. 1). Because di erent batches of eggs may develop at di erent rate, treatment timing was determined during the experiment based on observations of PB release in untreated controls. Cytochalasin B (0.67 mg L 1 ) was applied to the fertilized eggs at 8^10-min post fertilization (PF). The treatment was ended when in eggs in the control group: (1) Polar body 1 was released in 90% of the eggs, and PB2 began to release (T1); (2) PB2 was released in about 25^30% of the eggs (T2); (3) PB2 was released in about 70^75% of the eggs (T3) and (4) eggs began to enter mitosis I or the polar lobe began to form (T4) (Fig. 1). Cytochalasin B was removed by rinsing treated eggs on a 25-mm screen with dimethyl sulphoxide (DMSO) seawater (0.1%) three times and then with seawater three times. Eggs were resuspended in seawater and culture in 20-L buckets. Three replicates were produced with di erent sets of parents. Eggs from eight to ten females were used for each replicate. Eggs were pooled and fertilized with sperm from two to four males before being divided into control and four treatment groups. Parents and gametes The dwarf surfclam used in this study were collected from Rhode Island. Clams were conditioned at 21 1C and under intensive feeding for 3^4 weeks. Gametes were obtained by natural spawning with thermal stimulation or by dissection. Eggs were passed through a 60-mm screen and collected on a 25-mm screen. Sperm were ltered through a 25-mm screen to remove large tissue debris. Fertilization, treatment and culture were all conducted at 22^23 1C. Experimental design and treatment Larval culture Embryos were cultured at a density of 10^50 ml 1. Starting at 24-h PF, larvae were cultured at 10^ 20 ml 1 and fed daily with Isochrysis galbana at a density of cells ml 1. Culture water was completely changed every other day, and larvae were collected and washed gently on screens of proper size. For separate ploidyanalysis, D-stage larvae were separated from trochophores by using a 44-mm screen placed on top of a 25-mm screen. Larval survival was determined for all groups at each water change. After metamorphosis, M. lateralis spat were cultured in upwellers and then in trays contained in a well-aerated re-circulating system at a temperature of19^20 1C. Changes of culture water (approximately 1/5 of total volume) were performed at everyother day r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^1194

3 Ploidy determination The ploidy of larvae and juveniles was determined using ow cytometry (FCM) with a DAPI staining (10 mgml 1, solved in Tris bu er: 10-mM Tris, 146- mm NaCl, 2-mM CaCl 2,22-mMMgCl 2,0.005%bovine serum albumin, 0.1%Triton-X and 10% DMSO). Larvae (200^300) were pooled for FCM analysis, and juvenile clams were analysed individually. Data collection and analysis Fertilization levels were determined as the percentage of divided eggs at 2-h PF. The survival level was calculated as the percentage of survivors of fertilized eggs. The length (the longest dimension) of larvae was measured under a microscope. Data were analysed using the statistics software SYSTAT 10. E ects of treatment (or group) were tested by ANOVA.Percentage data were arcsine-transformed before analysis (Dixon & Massey 1983). Signi cance level was set at Po0.05 unless otherwise noted. Results The actual number of parents used for each replicate and treatment parameters observed in each group are presented in Table 1. Di erent batches of eggs showed di erent developmental rates and levels of synchronization and subsequently, treatment timing varied among the three replicates. Eggs for the rst replicate developed the fastest, and PB1 was released in the majority of the eggs at 30-min PF. Cytochalasin B treatment for thet1 group ended at 32-min PF. Development in the third replicate was the slowest and least synchronized, and the T1 treatment ended at 36-min PF. Eggs for the second replicate were intermediate between Replicates1and 3 in terms of development rate and synchronization. Table 1 Number of parents used, starting time and duration of cytochalasin B (CB) treatments applied for tetraploid induction by meiosis inhibition in Mulinia lateralis Replicate Parents (, <) Starting time (MPF ) CB duration (min) T1 T2 T3 T MPF, minutes post fertilization. Eggs under CB treatment appeared to be dehydrated, and the cytoplasm shrunk slightly leaving small cavities between the cytoplasm and the fertilization membrane especially at the site of PB release. In over 40^60% of the treated eggs in the T1 groups, PB1 was clearly visible during the CB treatment, and PB2 was absent. In some eggs, no PB was observed before and during the CB treatment. In T4 groups, the majority of treated eggs (495%) released no PBs during the CB treatment. After the CB treatment, only a few eggs (o3%) in T4 groups released one or two PBs. In the T2 and T3 groups, variable proportions of eggs released no or one PB. The number of eggs used, fertilization level and survival in control and four treated groups are presented in Table 2. The number of eggs used ranged from to for diploid controls, and from to for treated groups. Fertilization level was high in all groups. The average fertilization level was 99.0% for the control,96.5% for T1,96.3% for T2, 97.3% for T3 and 97.4% for T4 groups. There was no signi cant di erence in fertilization level among groups (P ). Larval survival varied considerably within and among groups. Survival to D-stage (Day 2) was higher in the control groups (25.5^ 91.6%) compared with treated groups (8.2^41.2%). Because of the large variation and the small number of replicates, the di erence was not statistically different (ANOVA, P ). It was obvious, however, that the majority of the larvae from the treated groups were abnormal and never reached D-stage. They stayed as trochophores and died within 5 days. Survival to Day 10 or just before metamorphosis was 46.4% for the control group, 21.4% for T1, 15.7% for T2,11.6% for T3 and 13.2% for T4 groups. Again, the di erence was not statistically signi cant (ANOVA, P ). For larvae that reached D-stage, growth was similar in the treated and control groups as measured by the shell length at di erent stages (Table 3). Larvae from the treated groups were more variable in size than that from the control groups. In a number of comparisons, larvae from the treated groups had larger standard deviations than that from the control groups, while the means were the same (Table 3). Flow cytometry analysis showed that larvae from control groups were all diploid as evidenced by the single diploid peak at Days 1 and 4 (Fig. 2a and b). There was no change in FCM peak patterns over time. Diploid peaks are located at channel numbers of 80^85, which are slightly lower than the diploid peak expected from the haploid sperm (Fig. 2c). Larvae in r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^

4 Table 2 Number of eggs used, fertilized level and survival in control and cytochalasin B-treated groups of Mulinia lateralis Group Replicate Total eggs ( 1000) Fertilization level (%) Cumulative survival (%) Day 2 Day 4 Day 6 Day 8 Day 10 Control Average T Average T Average T Average T Average Table 3 Larval size (mm) with standard deviation (SD) in control and cytochalasin B-treated groups in Mulinia lateralis Day 2 Day 4 Day 6 Day 8 Day 10 Group Replicate Length SD Length SD Length SD Length SD Length SD Control Average T Average T Average T Average T Average the treated groups had complicated ploidy compositions. Possibly due to the production of aneuploids and cell death, the FCM peaks in the treated groups were broad, overlapping and di cult to separate and quantify.we did not quantify the peaks to avoid possible misrepresentation of the data, but the di erence in gross ploidy composition among groups was obvious.when all larvae, normal D-stage and abnormal 1190 r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^1194

5 Figure 2 Flow cytometry histograms of Mulinia lateralis larvae from control and cytochalasin B-treated groups. The X- axis is the channel number (or DNA content) and they-axis is the cell count. (a) Control group at Day1; (b) control group at Day 4; (c) haploid sperm; (d, g, j and m) all1-day old larvae from T1^4 groups respectively; (e, h, k and n) normal 4-day old D-stage larvae fromt1^4 groups respectively; (f, i, l and o) abnormal 4-day old trochophore fromt1^4 groups respectively. trochophores, were analysed at Day 1, T1 groups showed a large tetraploid peak, along with a diploid and small pentaploid peaks (Fig. 2d). The T2 groups showed diploid, tetraploid and pentaploid peaks with approximately the same size/area (Fig. 2g). In T3 groups, the pentaploid peaks were mostly larger than the tetraploid peaks (Fig. 2j). In T4 groups, the tetraploid peaks were almost absent, and larvae were primarily pentaploid or diploid (Fig. 2m). At Day 4, D-stage larvae and trochophores were separated for FCM analysis. In all treated groups, the normal D-stage larvae were primarily diploids with perhaps a small proportion of tetraploids int1 groups (Fig. 2e, h, k and n for T1^4 respectively). The trochophores were mostly tetraploid and pentaploid (Fig. 2f, i, l and o, for T1^4 respectively). All trochophores had died by Day 8, and normal veligers were separated into large (4120 mm) and small (o120 mm) size classes. In the control groups, all larvae were diploid (Fig. 3b). In T1 and T2 groups, the large larvae were all diploid (Fig. 3c and e), while the small larvae were mostly tetraploid and diploid (Fig. 3d and f). In T3 and T4 groups, there were diploid and triploid peaks in both size classes, but no tetraploids (data not shown). Larvae started metamorphosis at Day 10 and completed by Day 14. All groups produced some post-metamorphosis spat. Juvenile clams, 3^8 mm in size, were individually sampled for ploidy determination by FCM between 1 and 2 months PF. The majority (81^100%) of the juvenile clams from the treated groups were diploids (Table 4). Variable proportions of triploids were observed, ranging from 0% to 19%. In one of T1 groups, three tetraploid clams (0.6%) were found among 478 juvenile clams analysed. No tetraploids were found in any other groups. Discussion In most bivalves, mature eggs are arrested at prophase or metaphase of meiosis I, and release PB1 and PB2 only after fertilization (Gilbert 1988). The eggs are tetraploid before fertilization. Theoretically, blocking the release of both PB1 and PB2 produces pentaploids (Cooper & Guo 1989); blocking PB2 produces triploids (Stanley et al. 1981); and blocking PB1 produces tetraploids, along with triploids and aneuploids (Stephens1989; Guo, Cooper, Hershberger & Chew 1992a; Yang et al. 2000b). r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^

6 Figure 3 Flow cytometry histograms of 8-day old (eyed) Mulinia lateralis larvae from control and cytochalasin B-treated groups. The X-axis is the channel number (or DNA content), and they-axis is the cell count. (a) Haploid sperm, (b) eyed larvae from the control group, (c and d) big and small larvae fromt1group respectively, (e and f) big and small larvae from T2 group. Table 4 Ploidy composition of juvenile clams sampled from groups treated with cytochalasin B for meiosis inhibition in Mulinia lateralis Ploidy composition (%) Group Replicate Age (days) Sample size Diploid Triploid Tetraploid T T T T This study con rms that PB1 inhibition produces tetraploids, while blocking both PB1 and PB2 produces primarily pentaploids. Our data clearly show that high percentages of tetraploids are produced by inhibiting PB1 (T1 groups). Longer treatments covering PB1and PB2 lead to increasing proportions of pentaploids. The majority of larvae from T4 groups are pentaploid. The production of tetraploids from PB1 inhibition was expected. It has been shown that PB1 inhibition 1192 r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^1194

7 produces tetraploids through separated bipolar segregations where the two groups of bivalents from meiosis I entered meiosis independently, and only one set of chromosomes are released as PB2 (Guo, Hershberger, Cooper & Chew1992b; Que, Guo, Zhang & Allen 1997; Yang, Que, He & Zhang 2000a). The observations where viable tetraploids are obtained by inhibiting PB2 (Allen et al.1994)orby inhibiting both PB1and PB2 (Scarpa et al.1993; Peruzzi & Guo 2002) are not explained or supported by the present study. The leading or most obvious explanation is that because of the unsynchronized nature of eggs development, treatments missed intended targets and actually blocked PB1. There are other possibilities also. First, it may be possible that after the retention of both PB1 and PB2 and depending on treatment duration, one set of chromosomes is lost inside the eggs leading to the formation of tetraploids. Secondly, longer treatments may have changed segregation pattern and led to the formation of true and viable viable tetraploids rather than the aneuploid^tetraploids produced from PB1 inhibition. Blocking PB1 is known to cause abnormalities in chromosome segregation such as tripolar segregation and uneven distribution of chromosomes, leading to massive aneuploidy (Guo et al.1992a,b; Que et al.1997;yanget al. 2000a). Finally, it is also possible that pentaploids may revert to a viable form of tetraploidy through chromosome loss. It has been shown that triploid oysters can revert to diploids at low frequencies (Allen, Howe, Gallivan, Guo & DeBrosse 1999). Our data support the leading hypothesis that tetraploids are produced by unintentional inhibition of PB1. TheT1 treatments intended for PB1 inhibition produced the highest proportion of tetraploid larvae at Day1, among D-stage larvae at Day 4, among eyed larvae at Day 8 and viable tetraploids at juvenile stage. Few tetraploid larvae and no viable tetraploid clam were produced by T4 treatments, which inhibited PB1and PB2. It is clear that most of the tetraploids cannot survive to D-stage. Even for the tetraploids that passed D-stage, their growth was retarded compared with diploids. Tetraploids have limited viability in most bivalve species studied (Diter & Dufy 1990; Guo, Hershberger, Cooper & Chew 1993; Guo et al. 1994). One hypothesis attributes the limited viability of tetraploid to a de ciency in cytoplasm (or cell number) due to the cleavage of a normal egg with a larger nucleus (Guo 1991; Guo et al. 1994). The fact that large numbers of tetraploids are produced using large eggs from triploids seems to support the cytoplasm de ciency hypothesis. However, there is mounting evidence that viable tetraploids can be obtained by direct induction using eggs from diploids (Scarpa et al.1993; Allen et al. 1994; Yang et al. 2000b; Peruzzi & Guo 2002). This study provides another example of production of viable tetraploid by direct induction. Because of the small number of tetraploids obtained (fewer than ve in most studies), the production of large numbers of tetraploids by direct induction remains a challenge. Further studies are needed to improve the e ciency of tetraploid induction from diploids. Future research should target meiosis I or mitosis I, rather than inhibiting both polar bodies. Acknowledgments We thank Dr Stefano Peruzzi for assistance in the lab and Dr Timothy Scott for providing clam broodstock. This work is supported bya grant from the NewJersey Sea Grant Consortium (R/BT-2001) and by a grant from New Jersey Commission on Science and Technology s R&D Excellence Program (No ). This is Publication IMCS/NJAES and NJSGC References Allen S.K. Jr & Downing S.L. (1986) Performance of triploid Paci c oysters, Crassostrea gigas (Thunberg). I. Survival, growth, glycogen content, and sexual maturation in yearlings. Journal of Experimental Marine Biology and Ecology102, 197^208. Allen S.K. Jr, Shpigel M.S., Utting S. & Spencer B. (1994) Incidental production of tetraploid Manila clams, Tapes philippinarum (Adams and Reeve). Aquaculture128,13^19. Allen S.K. Jr, Howe A., Gallivan T., Guo X. & DeBrosse G. (1999) Genotype and environmental variation in reversion of triploid Crassostrea gigas to the heteroploid mosaic state. Journal of Shell sh Research18, 293 (abstract). Allen S.K. Jr, Erskine A.J., Walker E. & Zebal R. (2003) Production of tetraploid Suminoe oysters C. ariakensis. Journal of Shell sh Research 22, 317 (abstract). Arai K.F., Naito F. & Fujino K. (1986) Triploidization of the Paci c abalone with temperature and pressure treatments. Bulletin of thejapanese Society of Scienti c Fisheries 52,417^422. Beaumont A.R. & FairbrotherJ.E. (1991) Ploidy manipulation in molluscan shell sh: a review. Journal of Shell sh Research10,1^18. Cooper K. & Guo X. (1989) Polyploid Paci c oyster produced by blocking polar body I and II with cytochalasin B. Journal of Shell sh Research 8, 412 (abstract). r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1187^

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