Growth and survival of reciprocal crosses between two bay scallops, Argopecten irradians concentricus Say and A. irradians irradians Lamarck

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Aquaculture 272S1 (2007) S88 – S93 www.elsevier.com/locate/aqua-online

Growth and survival of reciprocal crosses between two bay scallops, Argopecten irradians concentricus Say and A. irradians irradians Lamarck Haibin Zhang a , Xiao Liu a , Guofan Zhang a,⁎, Chunde Wang b a

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Pec-Nord Inc., Bureau 305, 1363 Avenue Maguire, Sillery, Quebec, Canada G1T 1Z2

Abstract A complete diallel cross between two bay scallop populations, Argopecten irradians concentricus Say (M) and A. irradians irradians Lamarck (C), was carried out. Growth and survival were compared among hybrids and pure populations. No significant difference in the shell length was found among the four groups on the first day of D-larvae. On day 10, shell lengths of the two reciprocal crosses (CM, MC)(♀ × ♂) were significantly greater than those of the CC (141.97 μm) and MM (146.20 μm) groups, with the growth rate of the MC (156.14 μm) cross greater than that of the CM (155.35 μm) cross. Also, heterosis for survival was significantly larger than that for growth. Both maternal origin and mating strategy had significant effects on growth and survival throughout the whole larval stage. Heterosis was also observed in later spat and adult stages. On day 170, the mean shell length, shell height and total weight of the CM cross were significantly larger than those of the other crosses (P b 0.05). The results from this study indicate that hybridization between A. irradians concentricus and A. irradians irradians may be a promising way for genetic improvement of existing bay scallop brood stocks in China. © 2007 Elsevier B.V. All rights reserved. Keywords: Bay scallop; Argopecten irradians; Diallel cross; Heterosis; Maternal effect; Survival; Growth

1. Introduction The first cases of heterosis were reported in the early 20th century (Shull, 1908). Since then, cross breeding has been widely applied for the genetic improvement of animals and plants (e.g. Madalena, 1993; Crow, 1998; Li et al., 2001; Apostolov and Slanev, 2002.), although the underlying genetic mechanisms are still poorly understood (Griffing, 1990; Hedgecock et al., 1996). In molluscs, many reports exist on the application of heterosis ⁎ Corresponding author. Tel.: +86 532 82898701. E-mail address: [email protected] (G. Zhang). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.08.008

in the genetic improvement of molluscan brood stocks (Newkirk et al., 1977; Newkirk, 1980; Mallet and Haley, 1983, 1984; Newkirk, 1986; Hawes et al., 1990; Koehn, 1991; Manzi et al., 1991; Hu et al., 1993; Hedgecock et al., 1995, 1996; Bayne et al., 1999). The positive correlation between allozyme heterozygosity and growth and fitness has been established in some species of bivalve molluscs (Zouros and Foltz, 1987; Koehn, 1991; Sheridan, 1997). However, very few studies have been carried out on crossing different populations or subspecies in scallops. Cruz and Ibarra (1997) carried out reciprocal crosses between two catarina scallop populations (Argopecten circularis, Sowerby, 1835) and found

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that there was a clear maternal effect for survival, and some heterosis after 15 days for larval shell length. After being first introduced in 1982, bay scallop Argopecten irradians has become an important mariculture shellfish in China (Zhang et al., 1992). Since 1982, several additional introductions have been attempted from different sites throughout the USA. As a result, several cultured populations of bay scallop now exist in China. Among these, the M population A. irradians concentricus Say was originated from North Carolina, the north of its natural distribution. The original stocks were introduced from North Carolina in 1995 (Zhang et al., 2000). The Canadian population A. irradians irradians Lamarck was originated from Lunenberg, Nova Scotia of Canada. The original stocks were introduced from Massachusetts of USA in early 1980s. In 2001, Pec-Nord reproduced this population in its hatchery in Lunenburg and about 5000 resulted adults were transferred to China in March 2002. As these two populations were different in both morphology and geographic distributions, and also because of the random genetic drift in cultured population (Blake et al., 1997; Beaumont, 2000), it is possible that genetic differences may also exist between these populations, which may be potentially important for the genetic improvement in bay scallop brood stocks. In this paper, a cross between these two populations was carried out to determine if there was a significant heterosis effect on growth that may lead to the improvements in productivity over pure populations. 2. Materials and methods 2.1. Brood stocks Brood stocks of the A. irradians concentricus Say (M) population, which was originally introduced into China from USA in 1995, were collected from Laizhou, Shandong in 2003. The Canadian population of bay scallop A. irradians irradians Lamarck (C) was first introduced from Canada in 2002, and its F1 offspring were used in this experiment. All stocks were conditioned in the hatchery of Jinying Aquatic Development Co., Ltd (Qingdao, Shandong) following the method of Zhang (1992). The conditioning temperature varied from 4 °C at the beginning to 21 °C before spawning. All experiments were carried out in the Spring of 2003.

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muscle. Each scallop was then placed in a 1 l beaker filled with 23 °C seawater to induce spawning under close observation. Whenever a scallop was observed to spawn only eggs or sperm, the gametes were collected immediately, and the beaker was then well washed with fresh water and filled with new water to collect more gametes. The eggs were not collected if a scallop spawned both eggs and sperm simultaneously. After spawning, eggs were washed on a 25 μm screen. Eggs were sampled and checked under microscope for fertilization caused by sperm contamination, and eggs were discarded if they were contaminated. For each population, only the gametes from 10 individuals that spawned both eggs and sperm but at different times were used in the crossing experiments. This method was proved to be feasible in the hermaphroditic bay scallop (Sun et al., 2006; Qin et al., 2007). Fertilization started right after all the gametes were collected. Two reciprocal crosses (C♀ ×M♂ and M♀ ×C♂) were created by fertilizing mixed eggs from 10 scallops of one population (M or C) with mixed sperm from 10 scallops of another population (C or M). To create the intra-population crosses (C♀ ×C♂, M♀ ×M♂), 10 scallops from each population were divided into two subgroups of 5. For each population, the eggs from one subgroup of 5 were fertilized with the sperm from another subgroup so that no selffertilization would occur. The resulting D-larvae from the same population were pooled together. 2.3. Larvae rearing Twenty hours after fertilization, D-larvae were collected on a 60 μm mesh sieve, and reared in 30-l tanks. Three replicates were set up for each mating group. The initial larval density was 10 larvae/ml in each culture vessel and was maintained with same level by adjusting water volume. Water was completely changed once every two days. All the equipments used were treated with fresh water for 5 min to prevent contamination among groups. Water temperature was kept at 21–23 °C. All seawater used was sand-filtered and UV-sterilized. The larvae were fed with the micro-alga Isochrysis galbana at a concentration varying from 20,000 to 100,000 cells/ml per day at different larval stages. 2.4. Spat and adult culture When the eye-spots appeared, the spat collectors (polyethylene nets) were set into the larval tanks. Twenty days later, the collectors with spat were transferred into collector bags and hung on longline systems in the sea. The collectors were sorted and the retrieved spat were then put into large mesh bags. Three months later, all spat were transferred into 10-layer adult growout cages at a density of 30 scallops per layer.

2.2. Spawning and fertilization

2.5. Sampling

Fifteen ripe individuals (Stage IV, gonads were fully extended with no visible intestine) (Sastry, 1963) were chosen from each population and exposed to air for 15 min. To induce spawning, each scallop was injected with 0.1 ml of 0.02 mM serotonin (5-hydroxytryptamine, Sigma) into the adductor

During the larval stage, 30 larvae in each mating group were randomly sampled to measure average shell length on days 1, 4, 7, 10, and 30. Survival was estimated on the same day. In the nursery and adult grow-out stage, shell length, shell height and total weight were measured on days 110 and 170.

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2.6. Statistical analyses The shell length data were transformed to logarithms to ensure normality and homoscedasticity (Neter et al., 1985). Percent survival values were transformed to angular values to stabilize the variances of errors (Rohlf and Sokal, 1981). The differences in growth and survival among the groups were analyzed with one-way analysis of variance (ANOVA) followed by multiple comparison tests (Tukey). To determine the effects of egg origin (M vs. C dams) and mating strategy (intra- vs. inter-population crosses) on growth and survival, a two-factor analysis of variance model was used (Cruz and Ibarra, 1997): Yijk ¼ u þ EOi þ MSj þ ðEO  MSÞij þeijk where Yijk = mean length (or percent survival rate) of the k replicate from the i egg origin and the j mating strategy; u = overall constant; EOi = egg origin effect on shell length (or survival) (i = 1,2); MSj = mating strategy effect on length (or survival)(j = 1,21); (EO × MS)ij = interaction effect between egg origin and mating strategy; and eijk = random observation error (k = 1,2,3). Heterosis (H) was calculated following the method of Van Vleck et al. (1987) and Cruz and Ibarra (1997): Heterosisk ¼ ½ðF1  PÞ  100=P where F1 = mean shell length (or survival rate) of reciprocal crosses; P = mean shell length (or survival rate) of both pure populations (intra-population crosses). All statistical analyses were performed with SPSS 11.0, and significance for all analyses was set to P b 0.05.

Table 2 Percent survival means (%) and Standard deviation (SD) in the four experimental groups at days 4, 7 and 10 Group/day

4

CC CM MC MM Heterosis (%)

7 b

10 a

90.4 ± 3.8 96.6 ± 1.9b 93.5 ± 2.1b 78.7 ± 6.7a 12.41

59.2 ± 5.0 71.0 ± 0.6a 89.7 ± 8.5b 67.3 ± 12.9a 27.02

32.8 ± 7.2a 47.3 ± 4.0ab 52.9 ± 3.6b 34.3 ± 11.9a 49.19

shell length of the two reciprocal crosses (155.35 μm for CM and 156.14 μm for MC) was significantly greater than that of the intra-population crosses (141.97 μm for CC and 146.20 μm for MM) (P b 0.05). As the growth rate was considered, the order of the four groups was (MC, CM) N MM N CC, but MC was not significantly different from CM. The survival rates of the two pure populations and two reciprocal cross populations are shown in Table 2. On day 4, the survival rate of CC was significantly greater than that of MM (P b 0.05) while no significant difference was found between the two reciprocal crosses (P N 0.05). On day 7, the survival rate of CM was not different from that of CC and MM (P N 0.05). The survival rate of MC significantly outperformed all three other groups (P b 0.05). On day 10, the survival rate of MC was higher than those of pure crosses, but the difference among the survival rates of CC, MM and CM were not significant (P N 0.05). During the whole larval stage, the survival rate of MC was the highest and that of CC was the lowest. Both egg origin and mating strategy had significant effects on shell length and survival during the whole larval stage (Table 3). Heterosis of shell length and survival rate (%) for the two reciprocal cross populations are shown in Tables 1 and 2.

3. Result 3.1. Larval stage Shell length data at the larval stage are shown in Table 1. No significant differences in the average shell lengths were observed among the four groups on the first day of D-larvae. Starting from Day 7, the mean shell length of A. irradians concentricus Say (MM) was significantly larger than that of the Canadian population (CC) (P b 0.05). On day 10, the average

Table 1 Larval shell length (μm) and Standard deviation (SD) for the four experimental groups at days 1, 4, 7 and 10 Group/day

1

4

7

10

CC CM MC MM Heterosis (%)

94.3 ± 3.3a 93.8 ± 2.8a 93.5 ± 3.1a 93.6 ± 2.8a 0

127.9 ± 10.5b 126.7 ± 5.9b 126.8 ± 5.0b 123.0 ± 5.1a 1.02

133.0 ± 10.7a 144.6 ± 10.1c 139.6 ± 8.6b 140.5 ± 9.2b 3.90

141.97 ± 12.8a 155.35 ± 15.0c 156.14 ± 10.2c 146.20 ± 10.6b 8.10

Means with the same letter are not statistically different (P N 0.05).

Table 3 Analyses of variance showing egg origin (EO) and mating strategy (MS) effects for length and survival during the larval stage Source

1d EO MS EO ⁎ 4d EO MS EO ⁎ 7d EO MS EO ⁎ 10d EO MS EO ⁎

d.f.

Shell length

Survival

M.S.

P

MS

P

MS

1 1 1

0.000401 0.000218 3.92E-05

0.205 0.350 0.692

– – –

– – –

MS

1 1 1

0.004995 0.002192 0.005439

0.003 0.049 0.002

140.7821638 303.194 20.08215427

0.010 0.001 0.242

MS

1 1 1

0.00213 0.026441 0.035313

0.137 0.000 0.000

312.0886727 453.3757297 81.05312245

0.025 0.011 0.200

MS

1 1 1

0.005529 0.100804 0.002848

0.038 0.000 0.136

11.51605085 287.772464 4.588115894

0.479 0.006 0.652

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Table 4 Growth traits (shell length, SL; shell height, SH; total weight, WT) and heterosis in reciprocal and pure crosses at days 30, 110 and 170 Group

CC CM MC MM Heterosis (%)

30d

110d

170d

SL(mm)

SL(mm)

SH(mm)

SL(mm)

SH(mm)

WT(g)

0.57 ± 0.15a 0.77 ± 0.11b 0.74 ± 0.13b 0.68 ± 0.20ab 21.35

21.76 ± 4.03a 24.04 ± 5.02b 22.11 ± 2.66ab 21.09 ± 3.01a 7.70

21.67 ± 3.84ab 23.45 ± 4.75b 21.48 ± 2.67ab 20.47 ± 3.03a 6.62

36.82 ± 4.32a 45.21 ± 5.58b 38.63 ± 3.29a 38.08 ± 2.84a 11.94

36.23 ± 3.95a 44.14 ± 5.32b 37.91 ± 2.94a 37.08 ± 2.46a 11.92

10.44 ± 3.01a 13.48 ± 4.46b 11.13 ± 1.59a 10.93 ± 1.87a 15.13

Heterosis for shell length and survival rate was observed for the whole larval stage and the degree of heterosis increased with age. There was a distinct difference among traits; heterosis for survival rate (10%–60%) was far greater than that for shell length (1%–9%). On day 10, heterosis in MC was greater than that of CM for both traits. 3.2. Spat and grow-out Shell length of reciprocal crosses (CM, MC) was significantly larger than that of CC on day 30 (P b 0.05), and the differences among of CM, MC, MM was not significant (P N 0.05). Heterosis was 21.35%. The difference in shell length between the two pure populations was not significant (Table 4). Shell length and shell height of inter-population crosses were larger than those of pure populations on day 110, but the difference among CC, MM and MC was not significant (P N 0.05). The observed heterosis values were 7.70% (SL) and 6.62% (SH), respectively. On day 170, the mean shell length, shell height, and total weight in CM were significantly larger than those of the other groups (P b 0.05). The differences were not significant among MC, MM, and CC (P N 0.05).

4. Discussion Maternal effects form an important source of variation in early growth and survival. The young are subject to a maternal environment during the first stages of their life, and this influences the phenotypic values of many metric characters even when measured on the adults (Falconer, 1981). Maternal effects include cytoplasmic inheritance, maternal nutrition via the egg or pre- and post-natal feeding, imitative behaviour and interaction between sibs through the mother (Mather and Jinks, 1971). For mollusc species, the amount of energy reserves in the form of lipids has been shown to be a strong determinant of success of early stages of larval development (Cragg and Crisp, 1991). The accumulation of these energy reserves is a function of female genetic background, nutrition, and other environmental conditions during sexual maturation in their environ-

ments (Barber and Blake, 1991). Newkirk et al. (1977) reported a significant maternal effect on the survival of larvae up to day 6 in crosses of the oyster, Crassostrea virginica. In the present study, a significant maternal effect was observed for shell length and survival of larval. From the results on day 7, it seems that the eggs from the M population resulted in larger larvae than the eggs from the C population. That the maternal effect was not observed at the beginning of the larval phases but appeared in a later stage, agree with Mallet and Haley (1984). They proposed two possible alternative hypotheses, the physiological hypothesis and the cumulative hypothesis. In our case, the results may be because the energy reserves contained in the eggs of the two populations are different, which is one of the main factors in the early stages of larval performance (Cruz and Ibarra, 1997). Although the two populations in our experiment were cultured in the same environment in the hatchery for more than two months, the long-term effects of living conditions on their energy reserve cannot be eliminated. Significant differences between reciprocal crosses are common, e.g., in the Pacific oyster C. gigas (Hedgecock et al., 1995) and the American oyster C. virginica (Mallet and Haley, 1984). Nagler et al. (2000) suggested that the maternal effect, specifically engendered by maternal mRNA, might be an important constituent (see also Davidson, 1986; Bosworth et al., 1994). In this experiment, growth and survival of the two crosses were also different. The results suggest that the importance of examining the performance of both the reciprocal mating for all crosses before determining the male and female parent population is to be used in commercial seed production of bay scallop. There have been many cross experiments carried out in molluscs, and the results have not always been consistent. Gaffney and Allen (1993) and Allen et al. (1993) conclude that there was no unequivocal evidence for the existence of viable interspecific hybrids among Crassostrea species. Lannan (1980) compared larval survival within

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and between crosses involving seven Pacific oyster C. gigas lines that had been subjected to two generations of full-sib matings. On average, the survival rates of the pure larvae line were significantly higher than that of the outbred larvae. Beaumont et al. (2004) carried out crosses between individuals of mussels Mytilus edulis L. and M. galloprovincialis L. There were no significant differences in percent yield or percent normality between pure lines and hybrids. Hybrid and pure line veligers were grown for approximately four weeks at 10, 14 or 20 °C. Regardless of temperature, in general, hybrid larvae grew slower than either of pure lines. Mallet and Haley (1983) compared three unselected American oyster C. virginica populations and their two-way crosses, and the results showed that the mean live weight in the crosses differed from their respective mid-parent at 40 months, and the survival rate differed from 10 to 40 months of age. On average, the crosses showed heterosis for growth rate and survival, being superior to the mean of their parent lines. Hedgecock et al. (1995, 1996) made controlled crosses between two inbred lines produced by spontaneous hermaphrodites in the Pacific oyster C. gigas, and proposed that epistasis could be a significant cause of heterosis. Studies using crosses of bay scallop are still very scarce. Due to the hermaphroditic character of this scallop, the sperm and egg of one individual are released intermittently or simultaneously so that crosses are usually difficult to carry out. Cruz and Ibarra (1997) compared the larval growth and survival of two Catarina scallop populations and their reciprocal crosses. There was a significant maternal effect on the growth of reciprocal crosses from day 11 to 17, after which heterosis first appeared. In the present study, heterosis was found not only in the larval stages but also in adults and heterosis for survival was greater than heterosis for growth. The growth rate and the survival rate of two reciprocal cross groups were greater than that of the two pure populations. Especially, for CM which showed greater heterosis compared to the two pure populations. The amount of heterosis in a cross between two particular lines or populations depends on the square of the difference in gene frequency between the populations. If the populations crossed do not differ in gene frequency, there will be no heterosis while the heterosis will be greatest when one allele is fixed in one population and the other allele in the other population (Falconer, 1981). Thus, our results imply that the genetic differences may exist between these two populations and that the cross between the two populations may be useful in a bay scallop genetic improvement program. In conclusion, significant maternal effects exist at the larval stage, suggesting that the selection of stock and the

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