Comparative study on tetraploid induction of the Fujian oyster Crassostrea angulata utilizing three typical methods

doi:10.21203/rs.3.rs-2612536/v1

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Comparative study on tetraploid induction of the Fujian oyster Crassostrea angulata utilizing three typical methods

https://doi.org/10.21203/rs.3.rs-2612536/v1

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The sterility advantages of triploids make them paramount for oyster aquaculture. Current hatcheries for triploid industrial breeding utilize tetraploids as male parents, and thus tetraploids are a core asset for triploid oyster culture. The oyster Crassostrea angulata is the most productive oyster species in China, however, the optimal conditions for tetraploid induction have never been examined. In this study, we investigated the effectiveness of three induction methods (Method Ⅰ, 2n ♀ × 2n ♂ + inducer abbreviated as DD + ind; Method Ⅱ, 3n ♀ × 2n ♂ + inducer abbreviated as TrD + ind; Method Ⅲ, 2n ♀ × 4n ♂ + inducer abbreviated as DT + ind) and three inducers (salinity; cytochalasin B, CB; and 6-dimethylaminopurine, 6-DMAP) for tetraploid induction in C. angulata. The optimal induction conditions for the three induction methods were a CB concentration of 0.5 mg/L treated for 20 min, 0.75 mg/L CB for a constant induction treatment of 20 min and 6-DMAP at a concentration of 100 mg/L for 20 min, respectively. When the optimal induction conditions were applied to a single pair of oysters, the D-larval tetraploid rate for the three methods were 18.93%, 58.32% and 51.61%, respectively. The survival rates of the experimental groups were only 10.36%, 28.07% and 21.99%, respectively, at day 30. The tetraploid rates for Method Ⅰ and Method Ⅲ, although decreased significantly over time, still reached 11.81% and 21.99% at 30 days, and 8% and 16% at 180 days of age, respectively. Tetraploid rates for Method Ⅱ were 58.32% − 71.28% at the larval stage and 80% at day 180. In summary, in spite of very different induction rates, viable tetraploid C. angulata can be harvested in all three methods. Method Ⅱ (TrD + inducer) was regarded as the optimal method to induce C. angulata tetraploids, and the optimal induction condition was a CB concentration of 0.75 mg/L and treated for 20 min under this method.

Crassostrea angulata

Tetraploid

Optimal induction conditions

Diploid

Triploid

Triploid shellfishes have gained increasing commercial interest in global aquaculture production, due to their faster growth rate and greater resistance to adversity compared to diploids (Normand et al. 2009; Walton et al. 2013), as well as for their sterility which allows for all-year-round market availability and superior palatability due to the absence of glycogen depletion to generate gametes during the reproductive cycle (Brake et al. 2004; Piferrer et al. 2009; Jeung et al. 2016; Melo et al. 2020). Since Stanley et al. (1981) reported the first paper on triploid Crassostrea virginica induction, numerous studies have been conducted on various triploid mollusks, such as C. hongkongensis (Qin et al. 2019), C. gigas (Guo et al. 1996; Buestel et al. 2009; Ibarra et al. 2017; Li et al. 2022), Pinctada fucata martensii (Komaru and Wada, 1990), Haliotis discus hannai (Wang et al. 2022), C. virginica (Callam et al. 2016; Matt and Allen 2021; Bodenstein et al. 2023)d angulata (Zhang et al. 2022). Although physical or chemical methods, such as cytochalasin B (CB), 6-dimethylaminopurine (6-DMAP), temperature, hypotonicity, hyperosmolarity and caffeine, can be applied to produce triploids by inducing diploids × diploids crosses (Desrosiers et al. 1993; Gérard et al. 1994; Nell et al. 1996; Gérard et al. 1999; Barreto-Hernández et al. 2018; Kong et al. 2011), the triploid rate produced in such manner was unstable, with low larval viability, and involved the use of toxic chemicals in the animals for the market. Until Guo and Allen (1994b) discovered a method for yielding viable tetraploids, and thus producing so-called natural triploids (Guo et al. 1996) - a crossing between diploids and tetraploids to produce 100% triploid descendants. This theory-turned-reality study has motivated numerous experts to brainstorm for tetraploid induction methods.

To date, three recognized methodologies have been developed for eliciting tetraploid shellfish. The first methodology (Method Ⅰ, 2n ♀ × 2n ♂ + inducer abbreviated as DD + ind) for the induction of tetraploid shellfish was to inhibit the release of PB1 from diploid fertilized eggs, which was firstly proposed by Guo (1991). However, no viable juvenile tetraploid oysters were obtained via this method (Guo 1991; Guo et al. 1992a, b). The “cell-number defciency” hypothesis illustrated the failure to obtain viable tetraploids through inhibiting diploid fertilized eggs (Guo, 1991; Guo and Allen 1994b), which is predicated on the fact that a tetraploid cell is characterized with twice the nuclear material of a diploid cell. The abnormal nucleus/cytoplasm ratio presented in diploid cells would lead to abnormal mitosis and reduced cell numbers (Piferrer et al. 2009; Peachey and Allen 2016; Aileen Tan et al. 2017). However, viable and fertile autotetraploid C. gigas were successfully cultivated by the above means (Benabdelmouna and Ledu 2007, 2015), and they posited the oocyte size was not a limiting factor for the success of the induction to autotetraploidy. After that, another method (Method Ⅱ, 3n ♀ × 2n ♂ + inducer abbreviated as TrD + ind) for inducing tetraploid oysters was proposed by Guo and Allen (1994a), in which triploid eggs were fertilized with diploid sperm and the first polar body emission was inhibited. Viable and fertile tetraploids can be obtained in this way, including C. gigas and C. virginica (Peachey and Allen 2016; Guo and Allen 1994a; Li et al. 2021; Guo et al. 2002; Eudeline et al. 2000a). However, the poor fertility of triploid shellfish was a pressing problem that has to be considered in applying this method (Brake et al. 2004; Peachey and Allen 2016). The third method (Method Ⅲ, 2n ♀ × 4n ♂ + inducer abbreviated as DT + ind), called the complementary method, utilized diploid eggs fertilized with tetraploid sperms and then suppressed the release of second polar body to create tetraploids. Survivable tetraploid C. gigas can be generated via this method (McCombie et al. 2005; Benabdelmouna and Ledu 2015). However, how to obtain sufficient number of exploitable tetraploid parents becomes a challenge to apply this method.

In China, oyster farming plays an important role in the shellfish industry, contributing more than 36% of the total marine shellfish production, totaling 5.4 million tons in 2021. Three major species of oysters are cultured in China, namely C. gigas, C. angulata and C. hongkongensis. Of which, the Fujian oyster C. angulata with 2.07 million metric tonnes constitute 38% production of farmed oysters in China, which is mainly cultivated in Fujian Province, and fractionally existed in Guangdong and Guangxi provinces over the recent years (China Fishery Statistical Yearbook, 2022). Although this oyster is the most productive out of the three oysters, the drawbacks such as small size and bland taste have seriously affected its market value. Consequently, cultivating triploid C. angulata may be an effective means towards their improvement (Zhang et al. 2022). However, tetraploid induction of C. angulata is not well documented. The efficiency of tetraploid induction differs from species to species. Optimization trials would usually be required to identify which method would be most effective (Gérard et al. 1999; Peachey and Allen 2016; Yang et al. 2000; McCombie et al. 2005; Eudeline et al. 2000a).

In this study, the effects of three reagents (salinity, CB and 6-DMAP) and three induction methods (Method Ⅰ, DD + ind; Method Ⅱ, TrD + ind; Method Ⅲ, DT + ind) were examined to induce tetraploid C. angulata, and the optimal induction conditions were identified. Additionally, we also evaluated the effect of tetraploid induction in a single pair of oysters under optimal condition, and variation in ploidy at its larvae and juvenile stages.

Source oysters

Diploid C. angulata were collected from Putian, Fujian Province, China. Triploid and tetraploid oysters were induced in 2021 by inhibiting the release of PB2 and PB1, respectively, akin to the Pacific oyster (Gérard et al. 1999; Eudeline et al. 2000), with all triploids and tetraploids cultured in Rushan, Shandong Province, China (36.45 °N, 121.42 °E). Diploid, triploid and tetraploid oysters were collected and transported to the shellfish breeding station in Laizhou, Shandong Province (37.3 °N, 119.9 °E) in March 2022. All three broodstocks were artificially cultured with conditioning water (temperature: 24.0-25.0 ºC; salinity: 30 psu) until sexually maturity.

Preparation for fertilization

Putative triploids and tetraploids C. angulata were opened and examined for sex under the microscope. Female triploid oysters and male tetraploid oysters were sort out and a small piece of gill tissue of each oyster was taken and ploidy was verified by flow cytometry (FCM) (Allen, 1983). Genuine female triploids and male tetraploids were singled out for fertilization. At the same time, diploids were also dissected and the sexes were identified by microscopy. Due to the poor fertility of triploid oysters, the eggs collection of ten triploids was treated as one replicate. Moreover, only the round and dark colored eggs were retained. Eggs were obtained by “dry stripping” to realize synchronous development and consistent hydration times (Eudeline et al. 2000b; Allen and Downing 1992). Eggs collected from oysters of different ploidy were placed separately in 2L beakers and soaked in seawater at 25 ºC for at least 45 min, but no longer than 60 min prior to fertilization to promote final maturation (Peachey and Allen 2016). The water temperature was maintained at 25 ± 0.5 ºC from the time of fertilization to the completion of the treatment to maintain the best synchronicity between the eggs (Eudeline et al. 2000b).

Spermatozoa were obtained from diploids and tetraploids and examined under the microscope for density and activity. Only sperm from vigorous males were collected. Since the release of polar bodies depended significantly on temperature and salinity (Allen and Downing 1986; Eudeline et al. 2000b; Li et al. 2021). Thus, the water temperature and salinity were strictly controlled at 25.0 ± 0.5 ºC and 27.0 ± 0.5 psu, respectively, during the experiment.

Tetraploid induction

Optimal conditions for tetraploid induction in Method Ⅰ (DD + ind)

The optimal conditions for tetraploid induction under this method were identified according to the following experiments (Li et al. 2021; Benabdelmouna and Ledu 2015). (1) Egg from diploid oysters were collected and the density of each group was adjusted to 2.5 × 10⁶ per liter, the CB concentration gradient was set to 0.25 mg/L, 0.50 mg/L, 0.75 mg/L and 1.00 mg/L, the 6-DMAP concentration gradient was set to 50 mg/L, 75 mg/L and 100 mg/L, the salinity concentration gradient was set to 6 psu, 8 psu, 10 psu and 12 psu. When the first polar body (PB1) of the fertilized eggs emerged, the corresponding inducers were added to each group, and the induction duration was set to 20 min for all groups. The reagents were washed off immediately after the induction treatment. Then, all fertilized eggs were transferred to 100-L plastic buckets for incubation in micro-aerated seawater with water temperature at 25 ± 0.5 ºC and salinity at 27 ± 0.5 psu. After 24 h, the D-larvae of each group were collected for flow cytometry (FCM) to verify ploidy (Li et al. 2021). Optimal CB concentration, 6-DMAP concentration and salinity concentration were determined based on the tetraploid ratio. (2) The egg density of each group was adjusted to 2.5 × 10⁶ per liter. When the PB1 of the fertilized eggs emerged, induction was continued for 15 min, 20 min and 25 min, respectively, according to the optimal CB concentration, 6-DMAP concentration and salinity concentration determined in step (1). The reagents were washed off immediately after the induction treatment. Then, the ploidy of each group was verified after incubation. Optimal induction duration was determined based on ploidy data. Three biological replicates were set up for each experiment, and a 2N (2N ♀ × 2N ♂) control group was also established.

Optimal conditions for tetraploid induction in Method Ⅱ (TrD + ind)

The optimal conditions for tetraploid induction under this method were identified according to the following experiments (Guo and Allen 1949b; Peachey and Allen 2016; Eudeline et al. 2000a): (1) Eggs from triploid oysters were collected and the density of each group was adjusted to 2.5 × 10⁶ per liter, the CB concentration gradient was set to 0.25 mg/L, 0.50 mg/L, 0.75 mg/L and 1.00 mg/L, the 6-DMAP concentration gradient was set to 50 mg/L, 75 mg/L and 100 mg/L, the salinity concentration gradient was set to 6 psu, 8 psu, 10 psu and 12 psu. When the PB1 of the fertilized eggs emerged, the corresponding reagents were added to each group, and the induction duration was set to 20 min for all groups. Optimal CB concentration, 6-DMAP concentration and salinity concentration were identified according to the tetraploid ratio. (2) The egg density of each group was adjusted to 2.5 × 10⁶ per liter. When the PB1 of the fertilized eggs emerged, induction was continued for 15 min, 20 min and 25 min, respectively, according to the optimal reagent concentration determined from the above steps. The optimal duration of induction was determined. Each experiment involved three biological replicates, and a 2N (2N ♀ × 2N ♂) control group was also established.

Optimal conditions for tetraploid induction in Method Ⅲ (DT + ind)

The optimal conditions for tetraploid induction under this method were identified according to the following experiments (McCombie et al. 2005; Benabdelmouna and Ledu 2015): (1) Eggs from diploid oysters were collected and the density of each group was adjusted to 2.5 × 10⁶ ind/L The CB concentration gradient was set to 0.25 mg/L, 0.50 mg/L, 0.75 mg/L and 1.00 mg/L. The 6-DMAP concentration gradient was set to 50 mg/L, 75 mg/L and 100 mg/L. The salinity concentration gradient was set to 6 psu, 8 psu, 10 psu and 12 psu. When 30% of fertilized eggs appeared with the PB1, each group was induced with the corresponding drugs and all groups were treated continuously for 20 min. The optimal CB concentration, 6-DMAP concentration and salinity concentration were determined based on the ploidy constitution. (2) The egg density of each group was adjusted to 2.5 × 10⁶ per liter. When the PB1 emerged in 30% of fertilized eggs, induction was continued for 15 min, 20 min and 25 min, respectively, according to the optimal reagent concentration determined from the above steps. The optimal duration of induction was determined based on the ploidy constitution. (3) The egg density of each group was adjusted to 2.5 × 10⁶ ind/L. When 30%, 40% and 50% of the PB1 appeared in the fertilized eggs, the corresponding optimal induction time was treated according to the optimal induction concentration determined in the above steps. The optimal treatment time was determined based on the ploidy data. Three biological replicates were performed for each experiment. Moreover, a 2N (2N ♀ × 2N ♂) control group was also constructed.

Investigated the veridical induction effectiveness of Method I, Method Ⅱ and Method III

The poor fertility of triploid oysters resulted in optimizing the induction conditions using eggs that were not from the one oyster. Although we strictly selected well-developed triploid oysters, the synchronization of their egg development was not guaranteed to be completely consistent. Therefore, after exploring the optimal induction conditions under three methods, we applied them to a single pair of oysters to ensure maximum synchronization of oocyte development to unveil the authentic induction efficiency of the three methods. All eggs were obtained by “dry stripping”. Each experiment was replicated thrice and control groups (2N, 2N ♀ × 2N ♂; 2.5N, 3N ♀ × 2N ♂; 3N, 2N ♀ × 4N ♂) were set.

Larvae rearing

The larvae of a single pair of oysters obtained under the optimal combination of induction conditions were reared in 100-L polyethylene buckets according to the method of Li et al. (2011), that is, the density of larvae per group was controlled at 1 larvae/ml. The water was changed 1/3 once a day. Larvae were fed Isochrysis galbana, and appropriate amount of Platymonas subcordigoramis were added during the umbo-stage and eyed-stage. When about 30% of the larvae developed eyespots, cleaning scallop shells were suspended in the bucket as attachment substrate. After attachment, the larvae were temporarily reared in an outdoor pool for two weeks and then transferred to Rongcheng, Shandong Province, China (37.11 °N, 122.35 °E).

Data analysis

Data are shown as mean ± standard deviation (SD), and the SPSS 26.0 was utilized for all analytics. One-way ANOVA and multiple comparison Tukey test were performed for variability in cleavage rate, D larvae rate, tetraploid rate and survival rate. Differences in survival between control and experimental groups were analyzed by independent-samples Test. P < 0.05 was considered a statistically significant difference.

The cleavage rate was described as the percentage of cleaved eggs to total fertilized eggs. D-larvae rate was defined as the ratio of D-larvae to total fertilized eggs. Moreover, the induction efficiency index would be the multiplication of the D-larvae rate and the tetraploid rate (Li et al. 2011), and the optimal induction condition is the group with the highest induction efficiency index. The tetraploid rate of larvae and juvenile oysters was determined by flow cytometry (Allen, 1983; Li et al. 2021).

Optimal conditions for tetraploid induction in Method Ⅰ (DD + ind)

Although the tetraploid rates were significantly different, all three reagents in Method Ⅰ produced tetraploids under different concentrations and treatment durations (Table 1). The optimal conditions for induction of tetraploid oysters when using CB as inducer were at a CB concentration of 0.5 mg/L for 20 min of induction (Fig. 1), with cleavage rate, D-larvae rate, tetraploid rate and induction efficiency index of 55.54%, 53.70% ,14.22% and 7.62%, respectively. Although the highest tetraploid rate was achieved when the CB concentration was 0.5 mg/L and the induction was continued for 25 min with a value of 16.8%, the D larvae rate was significantly lower than the rate when the induction was continued for 20 min (20 min: 53.70 ± 1.72%; 25 min: 42.28 ± 2.06%). The maximum induction efficiency index was obtained at a concentration of 75 mg/L for 6-DMAP and 25 min of induction duration in values of 2.69% (Table 2). As for salinity as an inducer, the highest induction efficiency index was reached at 12 psu and 25 min of continuous induction with a value of 0.72%, and the tetraploid rate under this condition was 1.65% (Table 3). A negative correlation was observed between cleavage rate and D larvae rate with reagent concentration and induction duration when CB and 6-DMAP were utilized as inducers. In contrast, the cleavage rate and D-larvae rate gradually increased with increasing salinity concentration.

Optimal conditions for tetraploid induction in Method Ⅱ (TrD + ind)

Different types and concentrations of induction reagents and induction periods yielded different proportions of tetraploids in Method II (Table 1). When the PB1 emerged, the tetraploid rate and hatching rate at CB concentrations of 0.75 mg/L for 20 min of constant induction were significantly better than the other combinations which used CB as an inducer, with cleavage rate, D larvae rate, tetraploid rate and induction efficiency index of 43.98%, 45.83%, 56.19% and 25.78%, respectively. The cleavage rate and D-larvae rate decreased with increasing concentration and duration of treatment when 6-DMAP was given as an inhibitor (Table 2). The maximum induction efficiency index was recorded at a concentration of 100 mg/L and a continuous treatment of 25 min with a value of 19.67%. When the salinity concentration was set to 10 psu and the treatment was continuously for 20 min was the optimal induction condition for salinity as an inducer The D-larvae rate, tetraploid rate and induction efficiency index under this condition was 54.77%, 24.58%, and 13.48%, respectively (Table 3).

Optimal conditions for tetraploid induction in Method Ⅲ (DT + ind)

The optimal induction conditions for method III were continuously induced by 6-DMAP at a concentration of 100 mg/L for 20 min when 30% of PB1 appeared (Table 4, Fig. 1). The cleavage rate, D-larvae rate, tetraploid rate and induction efficiency index under this scenario were 66.84%, 67.60%, 35.46% and 23.96%, respectively. When the PB1 appeared in 30% of fertilized eggs, the percentage of tetraploids achieved by CB treating with a concentration of 0.50 mg/L for 15 min was significantly higher than the other combinations using CB as an inducer. However, when subjected to salinity inducer, the D-larvae rate and tetraploid rate were only 58.75% and 17.84%, respectively, even under optimal induction conditions when 30% of fertilized eggs appeared PB1 to be treated continuously with a salinity concentration of 12 psu for 25 min, which were lower than those under optimal induction conditions with CB and 6-DMAP as inhibitors.

Investigated the veridical induction effectiveness of Method I, Method Ⅱ and Method III

From the above experiments, we discovered that the optimized induction condition in Method I was a CB concentration of 0.5 mg/L treated for 20 min (Table 1). And for Method II, the optimal induction condition would be a CB concentration of 0.75 mg/L continuously for a constant induction treatment of 20 min (Table 1). In Method III, the ideal induction condition occurred when 30% of the fertilized eggs exhibited PB1 and were exposed to 6-DMAP at a concentration of 100 mg/L for 20 min (Table 4). Accordingly, the optimum induction conditions from each of the three methods were applied to well-developed a single pair of oysters. The induction efficiency indexes under three methods were 12.57%, 42.87% and 32.41%, respectively. And the tetraploid rates of each group were 18.93%, 58.32% and 51.61%, respectively (Table 5). The quantity of tetraploid oysters in Method I and Method III decreased by 37.63% and 57.38%, respectively, compared to the initial tetraploid rate at day 30. Moreover, the FCM revealed still 2.91% of 2.5N larvae surviving at 30 days of age (Fig. 2). However, the tetraploid proportion was increased with the growth of larvae in Method II, with a tetraploid rate of 71.28% at 30 days of age. The survival rates of all groups under the three methods decreased dramatically, with only 10.36%, 28.07% and 21.99% under the three methods at 30 days of age, respectively. The tetraploid rate was only 8% according to Method I at 180 days of age (Table 6), while the proportion of triploids and diploids was 70% and 22%, respectively. Moreover, the proportion of tetraploids according to Method III was 16%, with the largest proportion being triploids at 80%. While 43 detections were genuine tetraploids in 50 oysters in Method II, the tetraploid induction rate was significantly higher than the other two methods.

Different methods yield significantly different tetraploids percentages

In this study, regarding method I (DD + ind), viable tetraploid C. angulata were obtained from experimental groups with eggs from multiple individuals or a single individual under all three induction reagents (CB, 6-DMAP and salinity), and despite the fact that the tetraploid rate decreased significantly over time, survivable tetraploid oysters (8%) were still available at 180 days. The successful inducing of viable tetraploids utilizing Method Ⅰ in this study may be due to the lower concentration of the inducer and the shorter duration of induction (the optimal condition was a CB concentration of 0.5 mg/L for 20 min) resulting in relatively minimal toxic effects on the fertilized eggs (Benabdelmouna and Ledu 2015). Benabdelmouna and Ledu (2007, 2015) gained viable autotetraploid C. gigas using Method I as well. They argued that the size of diploid eggs was not a limiting factor in the successful induction of tetraploids. Furthermore, Method I was also successfully applied to the tetraploidization of T. philippinarum, C. belcheri, C. iredalei and Mytilus edulis (Allen et al. 1994; Aileen Tan et al. 2017; McCombie et al. 2009). However, many experts have attempted to induce tetraploids in other shellfish utilizing this method, including C. gigas (Guo et al. 1992a, b), C. hongkongensis, C. sikamea (Qin et al. 2021), Chlamys farreri (Yang et al. 2000) and Mulinia lateralis (Yang et al. 2006), but no viable juvenile shellfishes were successfully obtained, and they attributed the failure to obtain viable tetraploids by this method to the “cell-number deficiency” hypothesis (Piferrer et al. 2009). The Method Ⅱ (TrD + ind) utilized in this study was inhibiting the first polar body release after fertilizing eggs from triploids with sperm from diploids (Guo and Allen 1994a). Encouragingly, the rate of tetraploid induced by this method was significantly higher than that of other methods, and similar results were achieved in the induction of tetraploid C. gigas (Eudeline et al. 2000a). Many studies have considered that poorly fertile triploids restrict the large-scale implementation of this method (Brake et al. 2004; Peachey and Allen 2016; Piferrer et al. 2009), but the warm environment and the inherent characteristics of the oyster have greatly expanded the fertile triploids (Ibarra et al. 2017; Normand et al. 2008; Tan et al. 2021). Additionally, mated triploids may be unusually fecund (Jouaux et al. 2010; Matt and Allen 2021). However, utilizing Method Ⅱ to induce tetraploids has to consider the additional problem that intentionally selecting highly fertile triploid females act as mothers (Guo and Allen 1994a) and grandmothers (McCombie et al. 2005) to produce new tetraploid populations may entail the risk of producing triploid offspring with fertility comparable to, or even higher than, that of their triploid ancestors (Benabdelmouna and Ledu 2015). Method Ⅲ (DT + ind) as a complementary method to enrich genetic diversity in tetraploids was to use diploid females fertilized with tetraploid males and then inhibit the release of the PB2. The best induction condition in this study using Method III was when 30 percent of the fertilized eggs exhibited PB1 and for 20 min were exposed to 6-DMAP at a concentration of 100 mg/L. Although triploids accounted for 80% at 180 days, some viable tetraploids (16%) were still detected. This method was also successfully applied to the induction of tetraploid C. gigas (McCombie et al. 2005), where meticulously selected diploid females could spawn suitable eggs for the induction of tetraploids. Additionally optimal breeding habitat and careful cultivation of diploid egg-derived tetraploid larvae may be contributing to the success in obtaining viable tetraploid oysters (Benabdelmouna and Ledu 2015; Benabdelmouna et al. 2007; Peachey and Allen 2016). Although viable tetraploids can be obtained via all three methods. However, higher induction and hatching rates mean less input and more gain, both for industry and research. As a result of our study, we revealed that Method Ⅱ was the most efficient method for tetraploid induction - the tetraploid rate was high to 80% at day 180. And the optimal induction conditions under this method were a CB concentration of 0.75 mg/L and a continuous induction of 20 min.

The tetraploid rate was significantly affected with the inducer type, initiation time and induction duration

The three inducers utilized in this experiment were CB, 6-DMAP and salinity, which were widely used in inducing triploid and tetraploid shellfishes (Gérard et al. 1999; Ledu and McCombie 2003; Allen and Downing 1986; Nell et al. 1996; Guo and Allen 1994b; Eudeline et al. 2000a; Li et al. 2021). CB mainly affects the polymerization of actin (Maclean-Fletcher and Pollard, 1980), which is requisite for the formation of the cleavage furrow of the polar body (Allen et al. 1989). The effect on fertilized eggs can be virtually instantaneous, thus the window for treating fertilized eggs required to be strictly controlled (Peachey and Allen 2016). 6-DMAP acts as an inhibitor of protein phosphorylation and protein kinase activity (Dufresne et al. 1991). Many experts have been able to induce shellfish polyploidization using 6-DMAP with an efficiency comparable to that of CB (Peachey and Allen 2016; Nell et al. 1996; Gérard et al. 1999). Moreover, hypotonicity induces polyploidy potentially attributed to the disruption of energy metabolism caused by hypotonicity, which affects the microtubules and microfilaments formation (Wang et al. 2009), has been successfully applied to the induction of shellfish polyploids, such as tetraploid C. gigas (Li et al. 2021), triploid Patinopecten yessoensis (Wang et al. 2009) and triploid C. gigas (Kong et al. 2011). Despite the different mechanisms of inducer action, the induction rate, hatching rate and fertilization rate significantly decreased with increasing concentrations of CB and 6-DMAP and decreasing salinity concentration in this study. The heavier the inducer used, the more irreversibly toxic it is for the cells, thus affecting their normal physiological activities (Nell et al. 1996; Ledu and McCombie 2003). One study attributed the successful induction of diploid eggs to viable tetraploids to the low concentration of the inducer (Benabdelmouna and Ledu 2015). The starting time and the durations of the treatment directly influenced the release of the polar bodies (Nell et al. 1996; Piferrer et al. 2009). Treatment commenced too early and duration too long will inhibit both first polar body and partial second polar body emission resulted in the birth of aneuploid larvae (Eudeline et al. 2000a; Guo et al. 1992). However, initiating induction too late or for too short a duration will miss the window of polar body emission and fail to induce (Eudeline et al. 2000a; Li et al. 2021).

The egg sources significantly affected the tetraploid rate

Many experts strive to optimize the induction conditions, including the induction method, the reagent types, the initiation time and the treatment duration (Nell et al. 1996; Eudeline et al. 2000a; Peachey and Allen 2016; Guo and Allen 1994a; Benabdelmouna and Ledu 2015; McCombie et al. 2005). But their induction rate was not as high as expected. Some studies pointed out that the asynchrony of egg development makes it challenging to precisely capture the exact correct window for treatment, resulting in lower induction rates (Eudeline et al. 2000a). The eggs utilized in this study to investigate the actual induction effectiveness of the different methods were from a single oyster, and the eggs were retrieved using the ‘‘dry stripping’’ method (Allen and Downing 1992). Furthermore, the oysters were carefully selected - the eggs were sufficiently round, dark and abundant, and the sperm viability was the best of the best. Combined with the two details mentioned above, this study yielded viable tetraploids using all three methods, which was not achieved by others (Peachey and Allen 2016; Aileen Tan et al. 2017).

Significant variations in tetraploid rates and survival rates during the larval and juvenile periods

The survival rate of larvae under all three methods in this study decreased significantly, especially during the first 10 days under all three methods. This could be firstly due to the very high toxicity of the inducer, which severely deteriorated the larval survival rate (Ledu and McCombie 2003; Yang et al. 2000; Peachey and Allen 2016). Additionally, blocking PB1 has been known to induce aberrations in chromosome segregation, such as tripolar segregation and heterogeneous chromosome distribution, causing massive aneuploidy and reduced viability (Guo et al. 1992a). The tetraploid rates obtained in this study were decreased by 92% and 84% at 180 days utilizing Method I and Method III, respectively, in comparison to the first day after incubation. The significant decline can be explained by the tremendous toxicity of the reagent causing irreversible damage to the tetraploid larvae. However, the reagent treatment itself was only part of the explanation and may not be the essential explanation for the decreased survival of tetraploid larvae. Actually, the diploid and triploid larvae were viable after comparable reagent treatment in many other studies (Allen and Downing 1986; Gérard et al. 1994). Other than this explanation, the reduced survival rate of tetraploids may also be directly related to the tetraploids themselves (Benabdelmouna and Ledu 2015), the structure and number of tetraploid cells have changed, which may give rise to difficulties in the development and physiology of the larvae (Comai, 2005; Piferrer et al. 2009), and poor cytogenetic quality of tetraploids compared to their diploid or triploid predecessors lead to most oysters rapidly losing their tetraploid status and becoming mosaics and aneuploids (Benabdelmouna and Ledu 2015). Furthermore, the imbalance of gene expression in tetraploids could likewise affect larval development (Guo and Allen 1994a; McCombie et al. 2005).

Blocking PB1 in Method I significantly changed chromosome segregation in meiosis II, which, in specific cases, was accomplished through either “unmixed tripolar” or “segregated bipolar” figures, causing only one set of chromosomes to be released as PB2, these two specific segregation patterns formed a significant number of tetraploid larvae as well as diploid, triploid, and aneuploid larvae (Guo et al. 1992), making it plausible that the larvae in our study under Method I were mostly diploids and triploids at 180 days. Besides the above reason, suppression of PB1 or PB2 in diploid fertilized eggs also resulted in triploids (Gérard et al. 1999). The tetraploid rate increased with larval growth in Method Ⅱ in this experiment. Ploidy analysis revealed that utilizing Method Ⅱ yielded larvae only with 2.5n and 4n peaks. Massive death of aneuploids causes an increase in the tetraploid rate under this method over time (Eudeline et al. 2000a), analogous phenomena were also observed in the tetraploid induction of the “Haida No.2” line of C. gigas (Cheng et al. data unpublished). However, not all 2.5N oysters died, FCM analyses revealed that 2.5N were still alive (Guo and Allen 1994b; Eudeline et al. 2000a).

In this study, three different methods (Method Ⅰ, DD + ind; Method Ⅱ, TrD + ind; Method Ⅲ, DT + ind) combining three different reagents (salinity, CB, and 6-DMAP) were used to probe the optimal conditions for tetraploid induction in Fujian oyster C. angulata. The optimal induction conditions in Methods I, II and III were a CB concentration of 0.5 mg/L treated for 20 min, 0.75 mg/L CB for a constant induction treatment of 20 min and 6-DMAP at a concentration of 100 mg/L for 20 min, respectively. The optimal conditions identified were subsequently applied to a single pair of oysters, we found that survivable tetraploids were available for all three methods at day 180, with tetraploid rates of 8%, 80% and 16%, respectively. These results can be used to guidance the tetraploid induction of C. angulata.

Ethics approval

The present study was performed according to the standard operation procedures (SOPs) of the Guide for the Use of Experimental Animals of the Ocean University of China. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Ocean University of China.

Competing interests

The authors declare no competing interests

Funding

This work was supported by grants from the National Key R&D Program of China (2022YFD2400305), the China Agriculture Research System Project (CARS-49), and Shandong Province (2021ZLGX03, 2021LZGC027).

Author Contribution

Yuanxin Liang: Conceptualization, supervision, resources, project admi. Geng Cheng: Resources. Xianchao Bai: Resources. Jianmin Zhou: Conceptualization, supervision. Haining Zhang: Supervision. Yong Chi: Resources. Gaowei Jiang: Conceptualization. Chengxun Xu: Resources. Qi Li: Conceptualization, supervision, resources, project admi.

Data availability

The data that support the fndings of this study are available from the corresponding author

upon reasonable request.

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Table 1 The effect of Method Ⅰ (DD + ind) and Method Ⅱ (TrD + ind) on tetraploid induction of Fujian oyster Crassostrea angulata (inducer: CB).

Condition	Cleavage rate (%)		D larvae rate (%)		Tetraploid rate (%)		Induction efficiency index (%)
Condition	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ
CB (mg/L)
0.25	63.97±1.07^a	74.3±2.68^a	64.19±1.46^a	65.54±0.64^a	4.38±1.18^d	22.94±2.68^c	2.82±0.83^c	15.04±1.85^b
0.50	52.26±1.84^b	61.91±1.6^b	52.22±1.2^b	57.43±2.15^b	15.51±0.48^a	67.67±4.25^a	8.1±0.19^a	30.25±1.07^a
0.75	45.27±4.23^c	52.12±1.83^c	46.24±2.71^c	44.92±1.85^c	11.7±1.01^b	68.01±3.87^a	5.42±0.58^b	30.59±2.96^a
1.00	37.27±1.5^d	37.27±7.75^d	42.81±2.07^c	37.36±2.41^d	7.56±2.16^c	43.89±2.65^b	3.22±0.70^c	16.38±1.16^b
Induction duration (minute)
10	71.44±1.12^a	72.98±1.94^a	65.43±3.4^a	62.52±0.91^a	2.37±1^d	11.03±0.5^c	1.53±0.59^c	6.9±6.9^d
15	63.88±1.2^b	67.22±1.15^b	61.32±1.6^a	55.35±1.32^b	8.38±0.35^c	22.26±1.42^b	5.13±0.11^b	12.33±12.33^c
20	55.54±2.12^c	43.98±0.73^c	53.70±1.72^b	45.83±2.66^c	14.22±1.94^b	56.19±1.48^a	7.62±0.92^a	25.78±25.78^a
25	43.9±1.49^d	31.3±1.59^d	42.28±2.06^c	38.88±3.19^d	16.80±0.9^a	55.43±4.75^a	7.09±0.22^a	21.63±21.63^b
Control group	92.89±0.21	83.2±2.14	93.06±2.32	85.18±1.16	/	/

Different superscript letters in the same column indicated significant differences (P < 0.05).

Table 2 The effect of Method Ⅰ (DD + ind) and Method Ⅱ (TrD + ind) on tetraploid induction of Fujian oyster Crassostrea angulata (inducer: 6-DMAP).

Condition	Cleavage rate (%)		D larvae rate (%)		Tetraploid rate (%)		Induction efficiency index (%)
Condition	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ
6-DMAP (mg/L)
50	65.68±2.44^a	61.42±1.13^a	53.21±2.88^a	57.16±1.90^a	5.34±0.90^b	17.13±1.7^b	2.84±0.48^a	9.8±1.170^c
75	51.72±1.39^b	54.40±1.11^b	43.11±2.20^b	44.32±1.96^b	8.10±0.34^a	44.15±2.26^a	3.49±0.16^a	13.73±0.57^b
100	40.38±2.57^c	51.94±1.54^b	35.19±2.01^c	31.11±0.67^c	4.11±0.8^b	45.49±2.02^a	1.46±0.35^b	20.18±1.57^a
Induction duration (minute)
15	67.18±2.19^a	72.14±2.23^a	50.72±0.72^a	62.97±2.55^a	3.00±0.63^b	23.78±2.81^c	1.52±0.34^b	15.02±2.36^b
20	53.22±2.37^b	61.92±1.4^b	46.15±2.72^a	43.33±2.16^b	5.54±0.34^a	45.25±1.81^b	2.56±0.26^a	19.59±0.88^a
25	43.56±0.88^c	43.14±2.5^c	44.94±4.77^a	34.61±1.72^c	5.93±0.68^a	56.83±1.14^a	2.69±0.57^a	19.67±0.80^a
Control group	82.37±3.91	82.06±1.42	75.58±2.46	73.37±0.84	/	/

Different superscript letters in the same column indicated significant differences (P < 0.05).

Table 3 The effect of Method Ⅰ (DD + ind) and Method Ⅱ (TrD + ind) on tetraploid induction of Fujian oyster Crassostrea angulata (inducer: salinity).

Condition	Cleavage rate (%)		D larvae rate (%)		Tetraploid rate (%)		Induction efficiency index (%)
Condition	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ	Method Ⅰ	Method Ⅱ
Salinity (psu)
6	21.63±1.37^d	20.67±1.67^c	32.24±1.61^d	22.14±1.34^c	0.41±0.71^c	17.36±1.54^c	0.14±0.24^c	3.84±0.31^d
8	25.47±0.46^c	22.18±1.58^c	38.45±1.41^c	26.1±1.86^c	1.48±0.20^b	31.48±0.98^a	0.57±0.09^b	8.23±0.82^c
10	34.08±2.02^b	29.72±1.13^b	45.47±2.68^b	34.32±1.92^b	1.8±0.19^ab	33.92±0.34^a	0.82±0.07^b	11.65±0.76^a
12	42.59±2.94^a	33.21±2.54^a	56.37±3.18^a	44.75±3.99^a	2.43±0.47^a	23.6±2.06^b	1.36±0.26^a	10.51±0.08^b
Induction duration (minute)
15	34.25±1.42^a	67.34±1.64^a	53.53±2.89^a	63.11±2.87^a	0.66±0.12^b	13.57±1.93^c	0.35±0.04^b	8.53±0.83^b
20	27.62±0.91^b	62.17±1.75^b	43.45±1.51^b	54.77±1.88^b	1.48±0.2^a	24.58±2.06^a	0.65±0.11^a	13.48±1.5^a
25	20.58±1.63^c	56.99±1.87^c	43.23±2.37^b	46.63±1.31^c	1.65±0.22^a	17.94±1.75^b	0.72±0.12^a	8.36±0.74^b
Control group	82.37±3.91	82.06±1.42	75.58±2.46	73.37±0.84	/	/

Different superscript letters in the same column indicated significant differences (P < 0.05).

Table 4 The effect of Method Ⅲ (DT + ind) on tetraploid induction of Fujian oyster Crassostrea angulata (inducer: CB, 6-DMAP and salinity).

Condition

Cleavage rate (%)

D larvae rate (%)

Tetraploid rate (%)

Induction efficiency index (%)

6-DMAP

Salinity

6-DMAP

Salinity

6-DMAP

Salinity

6-DMAP

Salinity

CB (mg/L)

0.25

76.12±0.58^a

72.36±0.82^a

20.96±0.89^c

15.17±0.51^b

0.50

63.78±1.62^b

68.07±0.85^b

37.19±2.21^b

25.31±1.39^a

0.75

56.19±3.23^c

56.91±1.91^c

42.02±1.62^a

23.92±1.56^a

1.00

32.33±2.58^d

41.69±1.45^d

40.04±1.34^ab

16.69±0.52^b

6-DMAP (mg/L)

72.27±2.09^a

71.48±0.77^a

16.08±2.69^b

11.49±1.91^c

66.42±1.77^b

56.08±1.77^b

35.18±0.97^a

19.22±1.35^a

100

42.92±2.41^c

44.68±1.33^c

48.35±0.29^a

21.6±0.5^a

Salinity (psu)

23.95±1.84^b

25.81±1.14^d

0.00±0.00^d

0.00±0.00^c

26.5±2.76^b

29.05±1.21^c

11.07±1.35^c

3.20±0.25^b

31.77±1.33^a

33.84±2.66^b

15.81±0.99^a

5.36±0.71^a

34.25±1.76^a

41.06±0.8^a

13.57±1.13^b

5.57±0.46^a

Induction duration (minute)

66.83±1.45^b

67.66±2.39^a

47.57±1.23^a

67.67±1.81^b

65.68±0.44^a

55.66±2.52^a

35.82±2.18^a

22.41±1.57^a

16.53±2.2^b

24.25±1.89^a

14.72±1.1^a

7.77±1.02^b

51.24±1.08^c

54.9±2.17^b

43.9±0.85^b

54.43±0.66^c

61.17±1.77^a

48.51±1.33^b

37.36±2.44^a

24.92±2.18^a

23.55±1.93^a

20.33±1.11^b

15.25±1.43^a

4.28±0.67^c

47.4±1.64^d

42.04±1.72^c

33.23±2.61^c

48.2±1.5^d

45.07±4.3^b

36.36±2.88^c

34.14±1.78^ab

18.75±0.86^b

23.7±2.75^a

16.46±1.11^c

8.47±1.07^b

10.48±0.64^a

Initiation time

30% PB1

64.77±1.38^a

68.84±0.83^a

56.92±3.77^a

69.16±0.37^a

67.60±1.2^a

58.75±0.97^a

34.04±1.66^a

35.46±2.21^a

17.84±1.16^a

23.54±1.26^a

23.96±1.1^a

10.48±0.64^a

40% PB1

51.86±1.48^b

61.77±1.44^b

42.96±2.53^b

62.03±1.55^b

54.94±4.37^b

49.62±3.98^b

24.22±1.37^b

20.26±1.49^b

15.62±0.85^a

15.01±0.59^b

11.1±0.65^b

7.77±1.02^b

50% PB1

47.66±1.3^c

46.87±3.3^c

35.49±0.7^c

42.7±2.42^c

32.07±1.56^c

38.33±1.43^c

8.58±0.42^c

11.02±0.87^c

11.15±1.35^b

3.66±0.03^c

3.53±0.37^c

4.28±0.67^c

Control group

96.12±1.73

95.38±2.36

94.67±2.05

89.26±0.55

90.81±2.72

88.41±1.12

Different superscript letters in the same column indicated significant differences (P < 0.05).

Table 5 The effect of the optimal induction condition out of the three methods on the tetraploids rate of a single pair of oysters.

Method	Cleavage rate (%)	D larvae rate (%)	Tetraploid rate (%)	Induction efficiency index (%)
Method Ⅰ	55.95±2.72^b	66.42±2.84^b	18.93±1.46^c	12.57±1.03^c
Method Ⅱ	75.98±0.43^a	73.49±1.24^a	58.32±1.03^a	42.87±1.24^a
Method Ⅲ	73.65±1.52^a	62.85±2.54^b	51.61±1.37^b	32.41±0.53^b

Different superscript letters in the same column indicated significant differences (P < 0.05).

Table 6 The variations in ploidy proportional composition of Crassostrea angulata under the optimal induction conditions of three methods at day 180.

Ploidy composition	Method Ⅰ	Method Ⅱ	Method Ⅲ	2N	3N	2.5N
2N	11(22%)	4(8%)	2(4%)	50(100%)	0(0%)	6(60%)
2.5N	0(0%)	3(6%)	0(0%)	0(0%)	0(0%)	2(20%)
3N	35(70%)	3(6%)	40(80%)	0(0%)	50(100%)	2(20%)
4N	4(8%)	40(80%)	8(16%)	0(0%)	0(0%)	0(0%)

The 2.5N control group had a very high mortality rate, with only 10 oysters surviving at 180 days. The remaining groups had sufficient numbers of surviving oysters, so 50 oysters from each group (except the 2.5N control group) were selected for ploidy testing.

No competing interests reported.

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Editorial decision: Major revision
01 May, 2023
Reviews received at journal
17 Apr, 2023
Reviewers agreed at journal
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Reviewers invited by journal
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Editor assigned by journal
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Submission checks completed at journal
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First submitted to journal
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Comparative study on tetraploid induction of the Fujian oyster Crassostrea angulata utilizing three typical methods

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