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Broadening of the genetic basis of the Atlantic bay scallop Argopecten irradians after interspecific hybridization and backcrossing.

ABSTRACT The growth characteristics and morphological features of the Atlantic bay scallop (Argopecten irradians irradians Lamarck), Chilean scallop (Argopecten purpuratus Lamarck), F1 hybrids of these species, and the offspring produced via 2 cycles of backcrossing between hybrid populations and the Atlantic bay scallop were examined to determine the feasibility of broadening the genetic basis of the bay scallop. Bidirectional fertilization and successful hatching were observed in F1 hybrids. Notable heterosis (i.e., > 1% of the midparent values) was detected in growth and survival throughout the whole life span of the scallops. The F1 hybrids showed universal feminization, but with low fertilization and hatching rates observed in backcrosses with bay scallops (partial sterility). Hematoxylin-eosin staining of paraffin sections of the feminine gonads of the hybrids was performed in this study. The male gonad was found to be vestigial, without any active sperm, whereas the female gonad was well developed and presented abundant eggs with a normal appearance. Increasing fertilization and hatching rates were observed in the second generation of backcrosses between hybrid populations and bay scallops compared with the first backcrosses. At the same time, a growth advantage and low temperature resistance were regained in the offspring produced from backcrossed breeding. This study describes a promising way to broaden the genetic basis of bay scallops with the aim of improving the growth characteristics of cultured scallop stocks.

KEY WORDS: bay scallop, Chilean scallop, hybridization, partially sterile, backeross, Argopecten irradians

INTRODUCTION

The bay scallop Argopecten irradians was first introduced into northern China from the United States in 1982 (Zhang et al. 1986). Mariculture of A. irradians has become an important industry in China, with a massive annual production of approximately 200,000 t being recorded in the past (Guo et al. 1999), which has increased currently to approximately 1,000,000 t. Despite several reintroductions of the original US stocks to restock breeding populations in China (Li et al. 2000), the genetic quality of the scallops in the industry has declined year after year as a result of inbreeding depression (Blake et al. 1997).

The bay scallop was introduced legally into the Canadian Atlantic on two separate occasions, in 1978 and 1989, for the purpose of establishing a bay scallop fishery (Whyte et al. 1993). Some cultured bay scallops have spawned and survived during the coldest winters, even when low rates and small numbers were observed. A wild population adapted to low temperature eventually formed in Nova Scotia and on Prince Edward Island (Mackenzie & Clyde 2009). The Chilean scallop Argopecten purpuratus was distributed originally in Peru and Chile. It is one of the most important commercial species in its native habitat (Martinez et al. 2007). In 2009, these two species were introduced into China by the Dalian Yiqiao Marine Seeds Co., Ltd., in an attempt to improve the shellfish industry. However, it was found to be difficult for Chilean scallops to survive during the winter in northern China because they come from a subtropical environment. We considered it possible that hybridization of the two geographical populations of scallops might aid the scallop breeding industry via potential heterosis or broadening of the genetic basis of cultured stocks.

Potential applications for heterosis have been reported widely in agriculture (Falconer 1981, Hedgecock et al. 1995, Apostolov & Slanev 2002, Zhang et al. 2007). In aquatic animals, Hedgecock et al. (2007) found that crossing among elite inbred lines of the same species was the most common way to achieve heterosis, although the mechanism underlying this phenomenon remains unclear. Interspecific crossing is also an effective method for exploiting hybrid vigor in many aquatic animals, including molluscs (Menzel 1977, Zouros et al. 1992, Beaumont et al. 2004, Batista et al. 2007, Zhang et al. 2007), fish (Jansson et al. 1991, Hartley 1996, Jansson & Ost 1997, Fernando et al. 2004), sea urchins (Rahman et al. 2005), and crustaceans (Shokita 1978, Sankolli et al. 1982, Lin et al. 1988, Bray et al. 1990).

Most of these studies focused only on the fitness of inter-specific and intraspecific F1 hybrids, whereas the effects of genetic mechanisms are, typically, most pronounced in F2 and backcross hybrids (Lynch 1991). Backcrossing maintains the desired genetic complexes that are already present in adapted genotypes while allowing recombination between exotic and adapted genomes. Such genomic rearrangements may disrupt epistatic interactions that confer fitness in specific environments (local adaptation) as well as gene interactions that are independent of the environment. Thus, backcrossing could potentially broaden the genetic basis of a starting population to be used in a selective breeding program by combining beneficial alleles. Research addressing the success and fitness of backcross hybrids in animals is scarce (Arnold 1997, Edmands 1999).

In this study, we performed a reciprocal cross between populations of the two species described earlier and two cycles of backcrossing between the hybrid populations and the bay scallop, with the aim of broadening the genetic basis of Argopecten irradians for use in future selective breeding programs. We report achieving reliable interspecific hybridization between the two geographical populations, and note that the F1 hybrids between A. irradians and Argopecten purpuratus were partially infertile.

MATERIALS AND METHODS

Parental Scallops

The parental Argopecten purpuratus (P) and Argopecten irradians (C) to be used for F1 hybridization were provided by the DALIANYIQIAO Marine Seeds Co., Ltd. Samples of both populations were conditioned in a hatchery at the DALIANYIQIAO Marine Seeds Co., Ltd. (Dalian, China), according to the protocol of Zheng et al. (2004). The conditioning temperature for the P and C populations was increased gradually from 5-20[degrees]C. Both of the populations were induced to spawn at 20[degrees]C in April 2009. F1 hybrids and F2 backcross hybrids were raised and used as parental broodstock in 2 cycles of backcrossing.

Spawning and Experimental Design

F1 Hybridization

Fifteen mature scallops from each parental population, in which the gonad had developed to stage IV (Sastry 1963), were chosen randomly for each replicate of the experiment. A combined method was used to induce spawning (Zheng et al. 2004) during which we induced every hermaphroditic individual to release male and female gametes in succession. First, the scallops from each population were exposed to air for approximately 30 min. Next, 0.1 mL of 0.02 mM serotonin (5-hydroxytryptamine; Sigma) was injected into the adductor muscle of each specimen (Cruz & Ibarra 1997). Last, the specimens were exposed to thermal shock. Each scallop was placed in a separate 1-L beaker filled with sand-filtered seawater at 23[degrees]C. Approximately 20[degrees]40 min later, all the animals first released male gametes for approximately 30 min. There was an intermission period of approximately 10 min after the release of male gametes ended. At this time, the spawning scallops were removed from the beakers and rinsed 2-3 times with 23[degrees]C seawater. Each scallop was then placed individually into a 1-L sperm-free beaker filled with filtered seawater at the same temperature to allow the scallops to continue spawning. The female gametes were collected and washed with filtered seawater using a 30-[micro]m mesh sieve to reduce the possibility of uncontrolled fertilization. Sperm contamination was assessed microscopically, and contaminated eggs were discarded. Last, the eggs of 10 animals from each population were chosen for insemination for each replicate.

All the female gametes from each scallop were divided into 2 equal portions. One portion of the female gametes was fertilized using a mixture of male gametes from the 10 individuals of the second species of scallop, and the other portion was fertilized using a mixture of male gametes from the 9 other individuals of the same species of scallop. Zygotes with the same mating type were pooled together. A total of 4 distinct genetic groups were produced via these interspecific reciprocal crosses and are identified as follows, with the first letter being based on the sperm source: PP and CC were the purebred groups, and CP and PC were the hybridization groups. The experiment was replicated 3 times using a total of 30 parental scallops from each species. Each replicate of every experimental group was kept and data were collected separately until the scallop reached adulthood.

Two Generations of Backcrossed Breeding

The universal feminization observed in mature F 1 hybrids or F2 backcross hybrids made it more convenient for us to obtain pure eggs of hybrids using the same spawning methods described earlier. Eggs from the CP and PC hybrid groups were fertilized separately with sperm from bay scallops. Then, 2 backcross groups (BCP1 and BPC1) were generated, and the second-generation backcross groups BCP2 and BPC2 were produced subsequently with the eggs of the first-generation backcross hybrids and sperm from the bay scallops. The purebred bay scallops used in each backcross were retained as controls (CC1 and CC2, respectively). The experiments were replicated 3 times using a total of 500 parental scallops from each population in every generation. Each replicate of every experimental group was kept and data were collected separately until the scallop reached adulthood.

Development and Feeding

The fertilized eggs from each group were stocked in polyethylene buckets or cement tanks filled with filtered seawater at 22[degrees]C and a salinity of 30 ppt at a density of 20 eggs/mL. The buckets or tanks were agitated by a stirrer every 2 h to improve the development rate. Approximately 30 h later, the embryos had developed to D-stage larvae. The initial larval density in each bucket or tank was adjusted to 10 larvae/mL. Each bucket was aerated by a single central air outlet situated just above the bottom of the bucket. The culturing temperature was adjusted to 24[degrees]C, and the seawater was exchanged completely through a mesh sieve every day. The larvae were fed with Isochrysis galbana at a concentration of 2,000-10,000 cells/day as they grew. All the equipment used for the different groups was submerged in freshwater for at least 5 min to avoid contamination.

When the larvae had developed into eyed pediveligers, polyethylene nets were placed in each bucket to serve as a substrate for larval settlement and metamorphosis. Approximately 20 days postfertilization, the spat in each bucket had grown to 500-600 [micro]m in size. The spat, together with the substrate, were then transferred into a mesh bag and hung on a floating line in the sea. The mesh bags were changed as the spat grew. Three months after fertilization, the juveniles were transferred into grow-out cages. No culling was conducted during any of the procedures described.

Sampling and Measurements

Eggs that underwent cleavage were considered to be fertilized successfully. The fertilization rate was calculated as the percentage of eggs that underwent cleavage relative to the total number of eggs at 60 min postfertilization. The development rate was quantified as the proportion of D-larvae relative to the total number of fertilized eggs. The larval growth and survival rates were determined based on the average shell length (SL) of 30 randomly sampled larvae and the larval density of each genetic group from all replicates at 1, 4, 7, and 10 days postfertilization.

Juvenile growth was monitored by measuring the shell length of 30 randomly sampled juveniles at several time points. On day 165, the growth traits of adult F1 hybrids, including the SL, shell height (SH), shell width (SW), and total weight (Wt), were measured using calipers (accuracy, 0.02 mm) and an

electric balance (accuracy, 0.1 g). The adductor muscles of the parental stocks and the adult progeny of the 4 genetic groups were stored in 95% ethanol for genetic confirmation.

Histological Observations

Hematoxylin-eosin staining of paraffin slices of the gonads of feminized, mature F1 hybrids was performed according to the method of Ren et al. (2007). The pathological changes in the gonad during sexual maturity were examined.

Genetic Confirmation

DNA from 2 parental populations and 5 hybrids was extracted from ethanol-preserved samples using the Fast200 DNA extraction kit (FASTAGEN, Shanghai, China) according to the manufacturer's instructions. Genetic confirmation of hybrids was conducted using the internal transcribed spacer 1 (ITS1) and ITS2 markers (Yu et al. 2001). The primer sequences used for ITS1 amplification were 5'GGTTTCTGTAGGT GACCTGCY (18S forward) and 5'CTGCGTTCTTCATC GACCC3' (5.8S reverse), and the primer sequences for ITS2 were 5'GGGTCGATGAAGAACGCAG3' (5.8S forward) and 5'GCTCTTCCCGCTTCACTCG3' (28S reverse). We prepared 25-[micro]L amplification reactions containing 1.5 mM Mg[Cl.sub.2], 0.2 mM dNTP, 0.2 [micro]M each primer, 20 ng template DNA, 1 U Taq polymerase, 2.5 [micro]L of 10x polymerase chain reaction (PCR) buffer, and 0.4 mg/mL bovine serum albumin. The PCR amplification protocol consisted of an initial denaturation at 95[degrees]C for 5 min, followed by 30 cycles of denaturation at 95[degrees]C for 1 min, annealing at 55[degrees]C for 1 min, and extension at 72[degrees]C for 1 min, with a final extension at 72[degrees]C for 5 min. All thermal manipulations were performed in a TaKaRa TP600 PCR Thermal Cycler Dice (TaKaRa, Japan). The amplified fragments were separated on 1.5% agarose gel containing 0.2 [micro]g/mL ethidium bromide, and were visualized using a UV transilluminator (BIORAD). The fragments of interest were excised and purified using the AxyPrep DNA Gel Extraction Kit (Axygen). The purified fragments from the 2 parental stocks were sequenced directly, whereas those from the hybrid individuals were cloned using the pEASY-T 1 cloning kit (Trangen, Beijing, China) and propagated in transl-T1 phage-resistant chemically competent cells (Trangen). The insert size was checked via colony PCR. The positive clones were sequenced for species identification. Six clones of the PCR products from each individual were picked randomly for sequencing to assay the 2 alleles of each locus.

Statistical Analysis

Shell length, SH, SW, and Wt were converted to logarithms, and the survival rates were arcsine transformed to increase normality and homoscedasticity before analysis (Rohlf & Sokal 1981, Neter et al. 1985).

Heterosis was calculated using the following formula (Cruz & Ibarra 1997):

% Heterosis = (F1 - P) x 100/P,

where F1 is the mean SL (or survival rate) in the reciprocal crosses and P is the mean SL (or survival rate) in both pure populations (intrapopulation crosses).

The differences in growth and survival among the progeny of the 4 groups were analyzed via multiple comparison tests using Tukey's HSD method with 1-way analysis of variance. The fixed effects of the mating type (intra- vs. interpopulation crosses) and the maternal effect (ME; P vs. C dams: egg origin) on growth and survival were examined using the following general linear model:

[Y.sub.ijk] = [mu] + [MT.sub.j] + [ME.sub.i] + [(MT x ME).sub.ij] + [e.sub.ijk],

where [Y.sub.ijk] is the mean length (or percent survival rate) of replicate k from egg origin i and mating type j, [mu] is an overall constant, [ME.sub.i] is the ME on SL (or survival; and i is P dam, C dam), [MT.sub.j] is the mating type effect on length (or survival; and j is intraspecific crosses, interspecific crosses); [(ME x MT).sub.ij] is the interaction effect between the ME and MT, and [e.sub.ijk] is the random observation error (k = 1, 2, 3).

Multivariate analysis of variance was performed to test the single and combinatorial effects of the MT (intra- vs. interspecific crosses) and the ME (P species vs. C species) on the means of most growth traits. All the statistical analyses were conducted using SPSS 17.0, and the significance for all analyses was set at P < 0.05.

RESULTS

F1 Hybridization

Fertilization and Larval Survival

Figure 1 lists the fertilization rates, development rates, and larval survival recorded in the experimental groups. Successful fertilization was observed in both the intraspecific and interspecific crosses. No significant differences (P > 0.05) were observed in fertilization rates or development rates among the 4 groups. On day 4, the survival rate in the PP group was significantly less than that of the other 3 groups (P < 0.05), and 18% heterosis was observed in the hybrid groups. On day 7, the survival rate in the PC group was significantly more than that of the purebred groups, whereas no statistically significant difference was detected between the CP group and the PC or the CC group. The PP group still presented the lowest survival rate among the 4 groups (P < 0.05), and the hybrid groups exhibited 30% heterosis. On day 10, the PC group continued to outperform the other groups significantly (P < 0.05), but no significant difference was detected between the hybrid groups (CP and PC) or the purebred groups (PP and CC; P > 0.05). During the entire larval period, the PC group and the PP group exhibited the highest and the lowest survival rates, respectively. The mating type had a significant effect on the survival rate during the entire larval stage, but the egg origin had a significant effect on the survival rate only on day 7 (P < 0.05; Table 1).

Larval and Juvenile Growth

The larval SL data are shown in Figure 2. The SL values obtained for larvae that developed from eggs of Argopecten irradians (PC, CC) were greater than those that developed from eggs of Argopecten purpuratus (CP, PP) on days 1,4, and 7 of the larval period. Moreover, significant differences in SL were observed on these 3 days among the 4 groups (P < 0.05), and the estimated heterosis values were 2%, 6%, and 7%, respectively, for these 3 larval stages. The average SLs of the hybrid groups (CP, PC) were still greater than those of the purebred groups (PP, CC) and showed 21% heterosis. Throughout the juvenile stage, the SLs of the hybrid groups (CP, PC) were significantly larger than those of the purebred groups (PP, CC; P < 0.05), and a high percentage of heterosis was also detected (Fig. 2). In the pure populations (PP, CC), the mean SL of the CC group exceeded that of the PP group significantly throughout the juvenile stage (P < 0.05). In contrast, SL did not differ significantly between the CP group and the PC group during the juvenile stage (P > 0.05), except for day 60 and day 90, when the SLs of the CP group exceeded those of the PC group significantly (P < 0.05). Both the ME and MT had significant effects on SL throughout both the larval and juvenile stages (P < 0.05; Table 2).

Harvest Traits

The harvest traits SL, SH, SW, and Wt were measured in samples from each of the 4 genetic groups (Table 3). No significant differences were detected between the purebred groups (PP and CC) for any of the traits examined or between the hybrid groups (CP and PC) for SH, SW, and Wt (P > 0.05). However, the values for all the traits in the hybrid groups were significantly greater than those in the purebred groups (P < 0.05). Multivariate analysis of variance revealed significant effects of the ME and MT, and their interaction on all the morphological traits examined (Table 4).

Genetic Confirmation

The parental populations and hybrid progeny were analyzed via PCR amplification of the ITS1 and ITS2 markers. All the samples examined produced a single band for ITSI at approximately 330 bp and a single band for ITS2 at approximately 500 bp, neither of which were distinguishable based on fragment size. Eight species-specific loci were found by sequencing the ITS1 fragments obtained from individuals from each parental population. Each of the 5 hybrid individuals exhibited both of the species-specific loci. Figure 3 presents the ITS1 sequences of a hybrid that matched the ITS1 sequence of Argopecten irradians (GenBank accession no. GU901162; e-value, 9e-167; identity, 99%) and the ITS1 sequence of Argopecten purpuratus (GenBank accession no. GU901163) based on BLAST analysis (e-value, le-160; identity, 99%).

Features of the F1 Hybrid

A developed byssus could be observed throughout the juvenile and adult stages in Argopecten purpuratus. Individuals of this species adhere either to the environment or to each other. In contrast, a byssus was found in Argopecten. irradians only in the juvenile stage and was degenerated in adults under the mariculture conditions applied in commercial farms in China. The hybrid populations (CP, PC) behaved more like their A. purpuratus parent and exhibited developed byssi.

Apparent feminization (Fig. 4A, orange part) of the primary hermaphroditic gonad (Fig. 4B; the female gonad is orange whereas the male gonad is white) was observed when the gonad matured. Only pure eggs, bare of sperm, were obtained when the feminized individuals were induced to spawn, and were abundant. The eggs were examined microscopically 2 h after spawning to confirm the absence of sperm. Hematoxylin--eosin staining of paraffin sections of the gonads of feminized hybrids (Fig. 4C, D) and bay scallops (Fig. 4E, F) was performed to check for pathological changes. The male gonads of feminized individuals were found to be degenerated, without any active sperm, whereas the female gonad was developed, showing abundant eggs with a normal appearance (Fig. 4F).

Backcrossed Breeding

Fertilization and Larval Survival

Figure 5 presents the fertilization rates, development rates, and larval survival recorded during the first cycle of back-crossing in the experimental groups. Partial infertility was observed in the backcross groups (BCP1, BPC1); approximately 3 million larvae developed successfully from 10 billion fertilized eggs, representing a particularly low development rate compared with the bay scallop control (P < 0.001). On day 4, the survival rates in the backcross groups were not significantly different from those of the bay scallop control group (P > 0.05). On day 7, the survival rates in the backcross groups were significantly greater than that of the control group, with a 6% survival advantage recorded. On day 10, the backcross groups still outperformed the control group significantly (P < 0.05), showing a 25% survival advantage. Figure 6 presents the fertilization rates, development rates, and larval survival observed during the second cycle of backcrossing in the experimental groups. Both the fertilization rates and development rates in the backcross groups were significantly less than those of the bay scallop control group (P < 0.05); however, an obvious increase in either the fertilization rate or development rate was observed compared with those recorded during the first backcrossing cycle. The larval survival rates in the backcross groups were significantly greater than that of the control group throughout the larval period (P < 0.05).

Shell Length Growth

The SL growth data obtained for the second cycle of back-crossing are shown in Figure 7. Throughout the experiment, except day 1, the SLs recorded in the backcross hybrid groups (BCP2, BPC2) were significantly greater than those of the CC2 control groups (P < 0.05), and an average growth advantage of 18% was detected during the second cycle of backcrossing.

DISCUSSION

Genetic improvement of commercial bay scallop stocks in China has always been of concern for scallop breeders. In past decades, reintroduction of stocks from America or Canada was conducted in an attempt to rejuvenate commercial cohorts, but the results were not sufficient in terms of the speed of inbreeding. Crossbreeding offers a possible alternative method for yield improvement. Most previous studies have focused on heterosis in bivalves with the aim of promoting the development of aquaculture via intrapopulation crosses between geographical populations or inbred lines (Mallet & Haley 1983, Mallet & Haley 1984, Hedgecock et al. 1995, Hedgecock et al. 1996, Cruz & Ibarra 1997, Zheng et al. 2006). Zhang et al. (2007) compared the growth and survival of 2 bay scallop subspecies (Argopecten irradians concentricus and Argopecten irradians irradians) and of intersubspecific reciprocal crosses between them. Significant heterosis and an ME (P < 0.05) on either growth or survival were detected throughout the life cycle of these scallops. These results suggested a promising method for genetic improvement of bay scallops. A number of successful interspecific crosses have been reported in molluscs (Menzel 1977, Zouros et al. 1992, Beaumont et al. 2005, Xu et al. 2009, Wang et al. 2011), but genetic improvement has seldom been realized.

Within the genus Argopecten, the bay scallop (Argopecten irradians Lamarck) and the Chilean scallop (Argopecten purpuratus Lamarck) were considered initially to be different species on the basis of their geographical distribution; A. irradians was first described along the eastern coast of North America whereas A. purpuratus was first reported in South America (Chile, Peru). In addition, A. purpuratus was classified into the Chlamys genus until chromosomal evidence placed this species in the genus Argopecten, rather than in Chlamys (Brand et al. 1990). The banding information obtained during chromosomal identification of the 2 species also provided clear taxonomic information (Gajardo et al. 2002, Wang & Guo 2004). The Canadian bay scallop population we used in this study was particularly adapted to cold temperatures because of its high-latitude distribution, as mentioned earlier. The complementary characteristics of these 2 populations could potentially lead to genetic improvement of cultured bay scallops. The F1 hybrids of the 2 populations were examined to assess the base population for later gene introgression. Two cycles of backcrossing between the hybrid populations and Canadian bay scallops were conducted to determine the feasibility of broadening the genetic basis of bay scallops.

F1 Hybridization

Our results demonstrate successful hybridization between a Chilean scallop population and a bay scallop population, with considerable hybrid vigor being observed, implying the existence of a more complex genetic basis. The 2 populations could be fertilized by each other using the technique of differential gamete isolation (Zheng et al. 2004). The PC group presented the highest fertilization and development rates, although no significant differences in these parameters were detected among the 4 genetic groups (PP, CP, PC, and CC). The hybrid groups showed significantly greater survival rates than the purebred groups (P < 0.05) throughout the larval stage. The heterosis value related to SL, which reached a peak of 43% on day 20, increased gradually during the larval stage and then declined during the juvenile and adult stages. The Wt exhibited the greatest hybrid vigor among the growth traits examined in this study. Overall, the heterosis observed during this study was in accordance with that obtained via hybridization of the hard clams Mercenaria mercenaria and Mercenaria campechiensis, which produce fertile F1 hybrids and show positive heterosis (hp > 1) in terms of their growth rates and tolerance to environmental variables (Menzel 1962, Menzel 1977). However, interspecific hybridization in other bivalves has led to different results regarding growth. For example, hybridization between the mussels Mytilus edulis and Mytilus galloprovincialis resulted in no positive heterosis in growth rates (Beaumont et al. 2004).

In the current study, the groups with the same egg origin showed similar growth rates before day 20, whereas the groups with different egg origins showed significantly different growth rates (P <0.05), which suggests a significant ME. Maternal effects are usually detected in viability and growth rates during early developmental stages (Schwabl 1996, Eising et al. 2001). This result is not surprising because the success of early development in mollusc species depends primarily on the amount of energy reserved in yolk materials (Cragg & Crisp 1991).

Heterosis and an ME were observed together during the first 20 days of the experiment. This finding is inconsistent with the results of former studies in which the ME always masked the expression of hybrid vigor at the beginning of the larval stage (Cragg & Crisp 1991, Cruz & Ibarra 1997). Although the larvae that developed from the eggs of the C population showed greater survival rates than those of the P population during the entire larval stage, a significant ME on larval survival was detected only on day 7. Hybridization between Argopecten irradians concentricus and Argopecten irradians irradians (Zhang et al. 2007) produced a similar result. The phenomenon of the ME not appearing until day 7 could be caused by the transition between the larvae's nonfeeding and feeding stages, coupled with different lipid levels in the eggs produced by different females (Mallet & Haley 1984). Although they were exposed to the same conditions for several months before spawning, the parents of the 2 species may have presented different egg energy reserves. This difference could result from different culture environments during early growth stages or from the nature of these populations, and this variation in egg energy reserves could lead to an ME (Cruz & Ibarra 1997). Another important process that could cause an ME is mitochondrial heredity because the mitochondria are passed to the offspring mostly through the egg (Cundiff 1972).

Reports of hybrid identification using molecular tools are still very scarce. Xu et al. (2009) confirmed hybridization between Crassostrea ariakensis and Crassostrea sikamea successfully using species-specific ITS1 sequences. In the current study, species-specific ITS1 and ITS2 sequences were amplified in the 4 groups of progeny and were assumed to differ in size between the 2 species. However, the small difference between the sizes of the ITS PCR products prevented us from distinguishing hybrids from purebreds. Therefore, we sequenced the ITS1 DNA fragments from the 2 purebred groups (PP and CC) and obtained 8 species-specific markers. Both of the species-specific ITS sequences from the 2 species were detected in each hybrid individual we examined, which confirms the success of the hybridization (Fig. 4).

Apparent feminization was observed when the gonad matured. As described in Results, the male gonads of feminized individuals were found to be vestigial, without any active sperm, whereas the female gonad was well developed and showed abundant eggs with a normal appearance based on the examination of hematoxylin--eosin-stained paraffin sections (Fig. 4C). This phenomenon was most likely associated with male sterility, which is common in hybrids between distantly related plants or animals (Kaul 1988, Luo 1990). Male sterility can be thought of as the product of a genetic conflict between 2 genomes that have different modes of inheritance. High rates of male sterility have emerged in attempts to exploit heterosis in hermaphroditic crops (Zhao & Wang 2005, Eva & Michiel 2002). This new characteristic caused by these interpopulation crosses could even alter the breeding mode in the bay scallop industry.

Backcrossed Breeding

As hermaphroditic animals, the bay scallop and Chilean scallop could not be crossed effectively on a large scale. In addition, the introduced Chilean scallop has failed to form a native population for use in continued future crosses because of environmental maladjustment. Backcross breeding is the reasonable way to achieve genetic improvement in the long term, although the eggs of the obtained hybrids were also found to show partial sterility.

Various theoretical and empirical studies in crops have demonstrated that introgression of unadapted genes associated with a low yield potential requires at least 1 backcross to the adapted parent to recover or increase the yield potential of the adapted parent (Dudley 1982, Cox & Frey 1984). In Venezuela, hybrid red tilapia (Oreochromis aureus X Oreochromis mossambicus X Oreochromis urolopis honorum) are being backcrossed to ancestral lines in an effort to improve their growth and body shape (FAO unpubl, report).

Two generations of backcrossing were conducted between hybrids and bay scallops in the current study to improve the genetic basis of fitness traits. Although the fertilization and development rates obtained from the backcrosses were quite low as a result of the partial sterility of the F1 or backcross hybrids, adequate numbers of backcross hybrids were obtained based on the abundant F1 hybrid parents. The successively developed backcross hybrid individuals exhibited a significant fitness advantage in terms of either larval survival or growth, in accordance with the F I hybrids. The most positive findings were the increased fertilization rates and development rates observed after the cycles of backcrossing. These results could indicate the potential to overcome the observed partial sterility and to improve valuable characteristics gradually.

The current study examined hybridization between Chilean scallops and Canadian bay scallops and reports partial sterility of the F1 hybrids obtained from the 2 populations and the feasibility of backcrosses for the purpose of broadening the genetic basis of commercial bay scallop stocks.

ACKNOWLEDGMENTS

We thank the DALIANYIQIAO Marine Seeds Co., Ltd., for its kind support and Chunde Wang for the work regarding introducing Chilean scallops. We also thank Wenhu Cong, Youtao Wang, Tao He, and Yang Jang for their help during the experiments; and Fei Xu, Juan Li, and Lin Guo for their assistance with molecular identification. This research was supported by the National High Technology Research and Development Program (863 program, 2012AA10A410,2010AA10A401), Mollusc Research and Development Center, CARS, the Taishan Scholar Program of Shandon, and the Taishan Scholars Climb Program of Shandong.

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TABLE 1.
Analysis of variance results showing the
effects of maternal effect (ME) and mating
type (MT) on survival during the larval
stage.

                 Survival

Source      df       MS    P value

Day 4
  ME         1    41.385     0.440
  MT         1   683.529     0.011
  ME x MT    1    52.211     0.388
Day 7
  ME         1   170.142     0.039
  MT         1   671.741     0.001
  ME x MT    1    49.108     0.223
Day 10
  ME         1    11.368     0.367
  MT         1   205.520     0.004
  ME x MT    1     6.989     0.475

MS, mean square.

TABLE 2.
Analysis of variance results showing the effects
of maternal effect (ME) and mating type (MT)
on shell length during the larval and juvenile
stages.

                     Shell length

Source      df       MS    P value

Day 1
  ME         1     0.029   <0.001
  MT         1     0.006   <0.001
  ME X MT    1    <0.001    0.153
Day 4
  ME         1     0.149   <0.001
  MT         1     0.054   <0.001
  ME X MT    1     0.010   <0.001
Day 7
  ME         1     0.617   <0.001
  MT         1     0.110   <0.001
  ME X MT    1     0.219   <0.001
Day 10
  ME         1     0.578   <0.001
  MT         1     0.717   <0.001
  ME X MT    1     0.271   <0.001
Day 20
  ME         1     1.890   <0.00I
  MT         1     2.928   <0.00I
  ME X MT    1     1.868   <0.00l
Day 30
  ME         1     0.001    0.475
  MT         1     0.113   <0.001
  ME X MT    1     0.050   <0.001
Day 75
  ME         1    37.941   <0.001
  MT         1   347.569   <0.001
  ME X MT    1     6.925    0.040
Day 120
  ME         1   114.700    0.005
  MT         1   996.480   <0.001
  ME X MT    1     4.332    0.578

MS, mean square.

TABLE 3.
The mean [+ or -] SD for each growth trait (shell length,
shell height, shell width, and total weight) at day 170 for
purebred genetic groups (PP and CC) and hybrid groups (CP
and PC) of the Canadian bay scallop Argopecten irradians and
the Chilean scallop Argopecten purpuratus.

Group/growth
trait                   SL (mm)                   SH (mm)

PP              50.42 [+ or -] 6.34 (a)   48.46 [+ or -] 5.84 (a)
CP              56.39 [+ or -] 3.85 (b)   53.61 [+ or -] 3.48 (b)
PC              59.80 [+ or -] 4.35 (c)   55.92 [+ or -] 4.21 (b)
CC              50.51 [+ or -] 4.91 (a)   48.25 [+ or -] 5.36 (a)
Heterosis (%)             16                        13

Group/growth
trait                   SW (mm)                   Wt (g)

PP              18.58 [+ or -] 2.43 (a)   21.39 [+ or -] 7.03 (a)
CP              22.87 [+ or -] 2.65 (b)   32.41 [+ or -] 4.71 (b)
PC              22.30 [+ or -] 2.24 (b)   32.33 [+ or -] 7.48 (b)
CC              18.75 [+ or -] 2.23 (a)   21.30 [+ or -] 5.82 (a)
Heterosis (%)             21                        52

SH, shell height; SL, shell length; SW, shell width; Wt,
total weight. Within the same column, means with the same
letter are not significantly  different (P > 0.05).

TABLE 4.
MANOVA results for all growth traits at day 170 (shell
length, shell height, shell width, and total weight.

Source     Wilks'    F value   df *     P
          [lamdba]                    value

ME         0.300      2.915    4.5    0.136
MT         0.126      8.633    4.5    0.018
ME x MT    0.274      3.317    4.5    0.110

ME, maternal effect; MT, mating type.
* Numerator, denominator.
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Author:Zhang, Shoudu; Li, Li; Wu, Fucun; Zhang, Guofan
Publication:Journal of Shellfish Research
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Date:Dec 1, 2013
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