Phenotypic traits of two boring giant clam (Tridacna crocea) populations and their reciprocal hybrids in the South China Sea

Aquaculture 519 (2020) 734890

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Phenotypic traits of two boring giant clam (Tridacna crocea) populations and their reciprocal hybrids in the South China Sea

T

Yuehuan Zhanga,c,d, Zihua Zhoua,b,c,d, Yanping Qina,c,d, Xingyou Lia,b,c,d, Haitao Maa,c,d, Jinkuan Weia,c,d, Yinyin Zhoua,b,c,d, Shu Xiaoa,c,d, Zhiming Xianga,c,d, Zohaib Noora,b,c,d, ⁎ ⁎ Jun Lia,c,d, , Ziniu Yua,c,d, a Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, China b University of Chinese Academy of Sciences, Beijing 100049, China c Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, China d Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou 510301, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Tridacna crocea The boring giant clam Crossbreeding Phenotypic traits Heterosis

The boring giant clam, Tridacna crocea, is distributed along coasts in the South China Sea, and has formed several geographic populations. These populations represent potentially different genetic resources for aquaculture, and the evaluation of their genetic and production characteristics is important. To determine these performance characteristics, complete diallel crosses between two geographical populations with about 600 km distance, Zhaoshu Island and Huangyan Island, were conducted using pooled gametes at an experimental station in Sanya, Hainan Island Province. Two intra-population crosses (ZZ and HH) and two reciprocal inter-population crosses (ZH and HZ) were carried out successfully three times. High fertilization rates and D larvae rate were observed in all the experimental groups, suggesting that there was no sperm-egg recognition barrier between the two geographic populations. Reciprocal hybrids had a higher survival rate than the pure populations, but with the exception of metamorphosis rate there were no significant differences between the inter- and intra-population crosses. Growth heterosis was evident in both the spat and adult stages, and was mainly influenced by egg origin and mating strategy in the adult stage. In view of the growth differences between the two pure populations' progeny, the Huangyan Island population's progeny was more suitable for giant clam aquaculture than the Zhaoshu Island population's progeny. Our results demonstrate that crossbreeding between the Huangyan Island and Zhaoshu Island populations of boring giant clams can produce considerable heterosis, which has promising prospects for application in the giant clam aquaculture industry in the South China Sea.

1. Introduction Crossbreeding usually refers to close crossing, which is a common strategy used to increase yield, quality, disease resistance and other desirable traits of plants and animals. Intraspecific crossbreeding can be divided into population, stock and line crosses (Zhang et al., 2017a). Not all cross-breading produces positive heterosis; however, crossbreeding sometimes can result in offspring with decreased resistance to the environment stresses, gamete incompatibility, hybrid weakness or non-viable hybrids (Zheng et al., 2006). Crossbreeding has played an important role in the genetic improvement of aquatic animals for the aquaculture industry (Todesco et al., 2016). For instance, population crosses in scallops (Cruz and

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Ibarra, 1997; Zhang et al., 2007; Zheng et al., 2011), oysters (Kong et al., 2017), abalone (You et al., 2009), etc.; stock crosses in scallops (Zheng et al., 2006; Wang and Li, 2010), oysters (Zhang et al., 2017a), abalone (Li et al., 2017), etc.; and line crosses in scallops (Wang and Côté, 2012), oysters (Hedgecock et al., 1995; Hedgecock and Davis, 2007; Rawson and Feindel, 2012; Yin and Hedgecock, 2019), abalone (Li et al., 2018), etc. have been performed. Most progeny from these crosses exhibited positive heterosis and improved performance. In giant clams, several studies have been conducted on interspecific hybridization between Tridacna clams, but no previous reports on crossbreeding between populations, stocks or lines of a single species (Alcazar, 1988; Militz et al., 2017, 2019). Several giant clam species have considerable genetic and phenotypic variation have been found

Corresponding authors at: South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China. E-mail addresses: [email protected] (J. Li), [email protected] (Z. Yu).

https://doi.org/10.1016/j.aquaculture.2019.734890 Received 7 October 2019; Received in revised form 17 December 2019; Accepted 18 December 2019 Available online 19 December 2019 0044-8486/ © 2019 Published by Elsevier B.V.

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collected sperm were filtered through a 25 μm mesh screen to ensure no eggs were present. The collected eggs were set aside for at least 30 min before they were inspected using a microscope. Given that Tridacninae are simultaneous hermaphrodites (Braley, 1992; Lucas, 1994), care was taken to collect sperm and eggs separately from each clam. The eggs were discarded if they were found to be fertilized. In total, the eggs or sperm of eighteen individuals from each population were used in this study.

between populations in many geographical locations around the world (Benzie, 1993; Benzie and Williams, 1995; Benzie and Williams, 1997; Benzie and Williams, 1998; Kochzius and Nuryanto, 2008; Hui et al., 2016, 2017). Therefore, population crosses in giant clams have the potential to produce offspring which exhibit heterosis and so may be useful in the aquarium market and aquaculture industry. Giant clams are also commercially valuable in the aquarium trade and flesh is a popular food (Mies et al., 2017). During the past few decades, the increase in demand for their adductor muscles as an ingredient in Asian gastronomy, and their shells for carving and for the preparation od seed used in the freshwater pear-farming industry have made giant clams highly valuable (Neo et al., 2017). All eight species of giant clam (subfamily Tridacninae) are found along the coasts of the South China Sea. Six species belong to the genus Tridacna (T. gigas, T. derasa, T. squamosa, T. maxima, T. noae, T. crocea) and two belong to the genus Hippopus (H. hippopus, H. porcellanus) (Neo et al., 2017). Each of these species can be divided into five geographical populations based on their areas of distribution: Hainan Islands, Xisha Islands, Zhongsha Islands Dongsha Islands and Nansha Islands in the South China Sea. The boring giant clam, T. crocea, is the smallest of the giant clam species and has a wide distribution and relatively high density (Neo et al., 2015a, 2015b). Large phenotypic differences were found by the authors between the boring giant clams near Zhaoshu Island (Xisha Islands) and those near Huangyan Island (Zhongsha Islands), about 600 km apart. For example, those from Huangyan displayed fast growth and bright mantle coloration, while hardiness and dimmed mantle coloration were characteristic of the clams from Zhaoshu. To evaluate the performance of progeny from the two pure populations and their reciprocal hybrids, a series of 2 × 2 factorial cross experiments were conducted between the two geographic populations. The phenotypic characters, including fertilization, survival and metamorphosis, and growth during the larval, nursery, and growout phases of the four groups of progeny were compared under conditions that mimicked the coast of the South China Sea.

2.2. Cross-fertilization For each population, eggs from three parents were pooled and then divided equally into two 5-L beakers. Prior to fertilization, eggs were checked to see if any uncontrolled fertilization had occurred. Unfertilized eggs were used for inter-population crosses. One beaker of eggs from each population was fertilized with the pooled sperm from three clams from Zhaoshu Island (Z) and the other with the pooled sperm from three clams from Huangyan Island (H), at a ratio of 10–15 sperm per egg. Thus, artificial crosses were performed with the following four different combinations: Z♀x Z♂(ZZ), Z♀x H♂ (ZH), H♀x Z♂ (HZ), and H♀x H♂(HH). A small number of fertilized eggs were sampled to evaluate fertilization success and survival to D-stage larvae. The remaining fertilized eggs were suspended in a 500-L tank at a density of 30–40 eggs/mL for the incubation. Zygotes developed in sand-filtered seawater maintained at temperatures between 28.6 and 29.7 °C and salinity of 33 ppt. The entire experiment was repeated three times using three sets of parents, with each set consisting of pooled eggs from three individuals and pooled sperm from three individuals from each population (Table 1). 2.3. Larval and juvenile rearing Thirty-six hours after fertilization, D-larvae from each cohort (ZZ, ZH, HZ and HH) were collected on a 60-μm sieve and reared separately in 1000-L tanks. The initial larval density was adjusted to 2 larvae/mL, and was maintained at this level by controlling the water volume. Larvae were fed on Isochrysis galbana for the first 5 days at a density of 3000 cells/mL/day and were soaked for two hours per day from day 6 to day 8 in seawater containing symbiotic algae (zooxanthellae) at a density of 30 cells/mL. Larval rearing tanks were provided with gentle aeration and exposed to natural sunlight reduced through transparent polycarbonate roof sheeting and 50% light transmittance shade-cloth; this reduced the daily-maximum photosynthetically active radiation (PAR) directly above larval rearing tanks to between 0 and 563.9 μmol s−1 m−2 (Dataflow Systems Pty Ltd. light logger) (Braley et al., 2018; Militz et al., 2017, Militz et al., 2019). The rearing water was maintained at 28.6–29.8 °C with a salinity of 33 ppt. Most larvae (≥90%) attached and developed secondary shells, feet, gills and symbiotic systems, reaching the juvenile stage on day 15. No substrate was used during the spat nursing stage. Larvae set within 7 days, and newly settled spat were reared in the tanks for another ten weeks with gentle aeration, water flow and 50% natural lighting. Spat were transferred to coral stone substrate when the shell length (SL) reached 3–5 mm, and were reared an additional four weeks in the 1000L tanks. At that time, they were transferred to an artificial raceway system with continuously circulating water when the shell length of spat reached 8-10 mm. Thus, spat were nursed until they were one year old. The rearing water was maintained at 25.6 to 30.0 °C with a salinity of 33 ppt.

2. Material and methods 2.1. Broodstock collection and spawning Adult boring giant clams used as broodstock were sourced from Zhaoshu Island (center of Xisha Islands) (N 16.968334, E 112.260940) and Huangyan Island (east of Zhongsha Islands) (N 15.160812, E 117.760336) in the South China Sea (Fig. 1). Broodstock were collected by freediving at these two locations, where densities of T. crocea are highest within the lagoonal system. Clams were held in insulated containers filled with seawater and transported by boat to the Hainan Tropical Marine Life Experimental Station at the Chinese Academy of Sciences. Broodstock from two populations were held separately in 2000 L raceways and provided with gentle aeration and a continual flow of sand filtered seawater, sourced from Sanya Bay, Hainan Island. The size of two population's parents were measured by electronic Vernier calipe and electronic balance (N = 125 for samples). Average shell length, shell height, shell width and whole weight were 65.00 ± 6.95 mm, 46.65 ± 4.71 mm, 31.88 ± 3.40 mm, 70.03 ± 22.30 g for the Zhaoshu Island population's, and 87.49 ± 8.24 mm, 65.28 ± 6.09 mm, 47.66 ± 6.03 mm, 173.86 ± 53.37 g for the Huangyan Island population's (Fig. 2, A-D; Fig. 3, A-B). When these brood stocks from the two populations matured, they were induced to spawn by exposure to air for 10 min followed by a temperature shock from 27.0 °C to 30.0 °C. Once spawning initiated, individuals were placed separately into 10 L plastic beakers filled with seawater at 30.0 °C. The clams were under close observation to enable collection of uncontaminated eggs or sperm. The eggs and sperm were abandoned if a clam released eggs and sperm simultaneously. The

2.4. Sampling and measurements The hatching index (cleaved rate and D larvae rate), survival rate (at days 7, 15, 90 and 360), and growth (egg diameter, D larvae size and spat size across the same days) of each group were determined according to Zhou et al. (2020). Survival rate at day 7 and day 15 were 2

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Fig. 1. Location of parental populations for the boring giant clam Tridacna crocea in the South China Sea. Two red dots represent Zhaoshu Island and Huangyan Island, Respectively. One back dot indicates the Zhongsha Islands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Parental size of two populations for the boring giant clam Tridacna crocea in the South China Sea. ZZ indicates Zhaoshu Island population, while HH indicates. Huangyan Island population. A, B, C, D indicate shell length, shell height, shell width and whole weight of parents, respectively. 3

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Fig. 3. Photos of parents and progeny for boring giant clam Tridacna crocea. A, Zhaoshu Island population's Parents. B, Huangyan Island population's Parents. C, Zhaoshu Island population's Progeny. D, Huangyan Island population's Progeny. E, Zhaoshu Island♀ × Huangyan Island♂ Progeny. F, Huangyan Island♀ × Zhaoshu Island♂ Progeny. The bar indicates 40 mm, and it applied to all samples in Fig. 3.

defined as the ratio of the number of individuals at different developmental stages to the number of D larvae. Survival rate at day 90 and day 360 were defined as the ratio of the number of individuals at different stages to the number of spat on day 30. At day 90, spat (shell length approx. 3–5 mm) from each of the three replicates within each group (ZZ, HH, ZH and HZ) were divided equally between 40 substrates (coral stone 16–18 cm in diameter), for a density of 30 individuals/substrate. That is, there were a total of 3600 spat on 120 substrates for each group, and a total of 14,400 spat on 480 substrates in the entire experiment. The substrates were cleaned monthly. As the spat grew, the density was reduced from 30 individuals/substrate to 15 individuals/substrate by removing randomly selected spat. Moreover, the dead clams were discarded at every substrate cleaning, and the density of each group was readjusted to maintain similar levels among the groups.

Table 1 Experimental design for the crossbreeding of the boring giant clam between Zhaoshu island and Huangyan island populations. Parents

Z1♂

H1♂

Z2♂

H2♂

Z3♂

H3♂

Z1♀ H1♀ Z2♀ H2♀ Z3♀ H3♀

ZZ1 HZ1 – – – –

ZH1 HH1 – – – –

– – ZZ2 HZ2 – –

– – ZH2 HH2 – –

– – – – ZZ3 HZ3

– – – – ZH3 HH3

ZZ and HH indicate Zhaoshu island and Huangyan island population's progeny, respectively. ZH and NZ indicate reciprocal hybrids between Zhaoshu and Huangyan island populations, respectively. The subscript number 1, 2, 3 denotes three replicates, and each replicate was conducted by pooled pair mating. Each replicate consisted of pooled sperm from three clams and pooled eggs from three clams of each population.

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2.5. Data analysis

3. Results

Differences in mean hatching index, survival rate and growth parameter between groups and replicates were analyzed using multiple comparisons using a two-way ANOVA (Fishers). To improve the normality and homoscedasticity, the hatching rate and survival rate were arcsine-transformed prior to analysis, and growth parameters were logarithmically transformed (base 10). All statistical analyses were performed using SPSS 18.0. P < 0.05 was considered statistically significant for all experiments, unless noted otherwise. Heterosis was analyzed in this study to evaluate hybrids for potential use in aquaculture during the first year. Mid-parental heterosis (H) was used (Zhang et al., 2007, 2017b; Wang et al., 2011) and calculated using the following equation:

3.1. Eggs and larvae No significant differences in mean egg size were found between the two populations. Mean egg diameter was 95.1 for the Zhaoshu Island population and 94.4 μm for the Huangyan Island population (Table 2). High fertilizations and D larvae rates (> 90%) were observed among all four groups, suggesting that there were no sperm-egg recognition barriers between the different geographic populations. The hatching rate was mainly affected by the interaction between the egg origin and mating strategy. 3.2. Survival

H (%) = [XF1 − (XZZ + XHH)/2] × 100/[(XZZ + XHH)/2]

On day 7, mean survival rates of larvae from all four groups exceeded 80%, with no significant differences among groups. The heterosis for the larval survival rate was 5.02%, and the single heteroses of the ZH and HZ offspring were 4.29% and 5.75%, respectively. Larval survival was affected by the mating strategy (Tables 2, 3). On day 15, the metamorphosis rates of the ZH and HZ larvae were 12.64% and 23.97%, while those of ZZ and HH were 10.82%, 18.31%, respectively. Mid-parental heterosis was 25.68%, and the single heteroses of the ZH and HZ groups were 16.82% and 30.91%, respectively. Rate of metamorphosis was primarily effected by egg origin (Tables 2, 3). From day 90 to day 360, the survival rates of all four groups of progeny were over 85%, with no significant differences among the groups. On day 360, mid-parental heterosis for survival was 6.18%, and the single heteroses of the ZH and HZ groups were 5.99% and 6.37%, respectively. After day 90, the survival ability of progeny was mainly affected by the interaction between egg origin and mating strategy (Tables 2, 3).

where XF1 indicates the mean phenotypic value (shell length, survival rate, etc.) of the reciprocal hybrids, while XZZ and XHH indicates the mean phenotypic value (shell length, survival rate, etc.) on the same day of the Xisha Island and Nansha Island populations' progeny, respectively. To determine the effects of the egg origin (Z vs. H) and mating strategy (homozygous vs. heterozygous crosses) on the survival and growth, a two-factor analysis of variance was used (Cruz and Ibarra, 1997; Zhang et al., 2007, 2017b), as follows:

Yijk = μ + EOi + MSj + (EO × MS)ij + eijk Here, Yijk is the mean shell length, wet weight or survival rate of the k replicate from the i egg origin and the j mating strategy. EOi is the egg origin effect on the shell length (wet weight or survival rate) (i = 1, 2). MSj is the mating strategy effect on the shell length (wet weight or survival rate) (j = 1, 2). (EO × MS)ij is the interaction effect between the egg origin and the strategy, and eijk is the random observation error (k = 1, 2, 3). Single-parent heterosis, the increase in performance over the purebred offspring of the maternal population, was calculated using the formula (You et al., 2015; Zhang et al., 2017b):

3.3. Growth Thirty-six hours after fertilization, the reciprocal hybrid D larvae were significantly larger than the D larvae of the pure populations, showing an obvious mating strategy effect (Tables 3, 4). At the end of the planktonic stage, a similar growth trend was observed with slight heterosis displayed, although there were no significant differences between groups (Table 4). On day 15, the shell length of all newly-formed spat was over 210 μm, and HZ progeny were significantly larger than the other three groups, with a single heterosis of 8.64% (Table 4). During the grow out stage from day 90 to 360, reciprocal hybrids were always larger than the pure population progeny, showing marked heterosis (Fig. 3 CeF). These traits were predominantly affected by the mating strategy and egg origin (Tables 3, 4). HZ progeny in particular were significantly larger than those of the other three groups, and ZZ

IHZ (%) = (XHZ − XHH)/XHH × 100 IZH (%) = (XZH − XZZ)/XZZ × 10 where IHZ, IZH indicates single-parent heterosis for the HZ and ZH offspring, respectively. XHH, XZZ, XHZ, XZH indicates the mean phenotypic value (shell length, survival rate, etc.), respectively.

Table 2 Hatching index, survival ability and metamorphism of two population progeny (ZZ and HH) and their reciprocal hybrids (ZH and HZ), as well as heterosis (H and I). Items

ZZ ZH HZ HH H(%) IZH(%) IHZ(%)

Hatching index

Survival rate (%)

Egg diameter (μm)

Cleaved rate (%)

D-stage (%)

Day 7

95.06 ± 2.31a – – 94.43 ± 3.00a – – –

98.85 97.10 98.26 98.41 – – –

92.12 93.40 92.87 93.44 – – –

83.49 87.07 88.85 84.02 5.02 4.29 5.75

± ± ± ±

0.52a 2.47a 1.08a 0.54a

± ± ± ±

2.27a 2.62a 1.43a 2.95a

Day15

± ± ± ±

3.39a 3.50a 4.33a 4.37a

10.82 12.64 23.97 18.31 25.68 16.82 30.91

Day 90

± ± ± ±

2.53c 3.41c 4.38a 3.60b

89.21 92.48 95.55 91.23 4.21 3.67 4.74

± ± ± ±

Day 360

4.12a 1.37a 1.34a 3.23a

85.43 90.55 92.67 87.12 6.18 5.99 6.37

± ± ± ±

1.37a 1.37a 1.37a 1.37a

X ± SD indicates mean ± standard deviation. H indicates mid-parent heterosis; and IZH and IHZ indicate the single parent heterosis of ZH and HZ groups, respectively. For cleavage rate, D-stage rate, cumulative survival on different days, n = 9 (3 replicates × 3) in each experimental group. Different superscript letters in each column indicate significant difference (p < 0.05). 5

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Table 3 Analysis of variance (ANOVA) showing the egg origin (EO) and mating strategy (MS) effects for survival and growth of each experimental group at different time points. Items

Day7

Day 15

Day 90

Day360

Source

EO MS EO × EO MS EO × EO MS EO × EO MS EO ×

df

MS

MS

MS

MS

Survival rate

1 1 1 1 1 1 1 1 1 1 1 1

Shell length

MS

F-value

p

MS

F-value

p

2.101 0.035 1.640 0.360 3.450 0.689 2.727 1.415 0.356 15.708 4.768 0.914

0.008 < 0.001 0.006 < 0.001 0.093 0.005 0.099 0.037 0.002 0.035 0.009 < 0.001

0.007⁎⁎ < 0.001⁎⁎⁎ 0.009⁎⁎ < 0.001⁎⁎⁎ 0.037⁎ 0.004⁎⁎ 0.046⁎ 0.023⁎ 0.003⁎⁎ 0.029⁎ 0.006⁎⁎ < 0.001⁎⁎⁎

0.559 0.874 0.940 7.631 0.978 23.452 8.166 8.921 30.171 7.289 0.936 9.247

< 0.001 < 0.001 < 0.001 0.003 < 0.001 0.013 0.074 0.067⁎ 0.249 0.045 < 0.001 0.087

< 0.001⁎⁎⁎ < 0.001⁎⁎⁎ < 0.001⁎⁎⁎ 0.002⁎⁎ < 0.001⁎⁎⁎ 0.027⁎ 0.039⁎ 0.036⁎ 0.127 0.038⁎ < 0.001⁎⁎⁎ 0.075

⁎

Indicates p < 0.05. Indicates p < 0.01. ⁎⁎⁎ Indicates p < 0.001. ⁎⁎

Williams, 1998). Genetic divergence has been shown to exist between geographically isolated populations of most species of giant clam, including T. gigas (Benzie and Williams, 1995, Benzie and Williams, 1996), T. squamosa (Hui et al., 2016), T. maxima (Benzie and Williams, 1997), T crocea (Hui et al., 2017). However, since giant clams are hermaphroditic and may therefore self-fertilize, genetic diversity and heterozygosity within populations may be lacking (Hui et al., 2016, 2017). Therefore, there is great potential for population crosses to produce hybrid offspring with considerable heterosis which could be used to increase the efficiency of giant clam aquaculture in the South China Sea.

progeny were the smallest of the four groups. On day 360, the mean shell lengths of the ZH and HZ progeny were 45.06 mm and 51.28 mm, respectively, and the mean whole weights were 14.87 g and 19.28 g, respectively, compared with 38.84 mm and 10.96 g for the ZZ progeny and 43.65 mm and 13.35 g for the HH progeny. Thus, the mid-parental heterosis was 16.79% for shell height and 40.52% for wet weight. The single heterosis of ZH was 16.01% for shell length and 35.68% for wet weight, while that of HZ was 17.48% for shell length and 44.49% for wet weight (Fig. 3, C-F; Table 4).

4. Discussion 4.1. Population differences

4.2. Heterosis

Phenotypic differences between the two pure populations were clearly evident in this study; the Huangyan Island population's progeny had more rapid growth and a higher rate of metamorphosis than that of the Zhaoshu Island population. At day 360, the mean shell length of the HH progeny was 12.38% longer and the mean wet weight was 21.81% heavier than those of the ZZ progeny. At day 15, the HH group's rate of metamorphosis was 69.22% higher than the ZZ group's. Considerable differences between the two populations are not unexpected, since they are geographically isolated by an archipelago barrier, the Zhongsha Islands (Fig. 1). Thus, reproductive isolation resulting from a barrier between populations is known to result in genetic differentiation as a consequence of natural selection, adaptation to environmental conditions, and genetic drift (Cruz and Ibarra, 1997). Therefore, information on the genetic population structure of giant clams is important for the design of breeding programs and the understanding of ecological and evolutionary processes (Benzie and

Crosses between genetically differentiated populations are expected to increase heterozygosity, reduce effects of recessive lethal genes, and enhance fitness, resulting in heterosis or hybrid vigor (Charlesworth, 2018). The heterosis of the hybrid progeny in this study had the following characteristics: (1) there was positive heterosis in terms of survival and growth throughout the entire experimental period; (2) the magnitude of heterosis varied between traits and life history stages; and (3) heterosis of reciprocal hybrids is asymmetric due to mating strategy. Heterosis for a single-locus, two-allele trait in a cross between two lines, stocks or populations is proportional to the square of the difference in gene frequency between the lines, stocks or populations crossed (Falconer and Mackay, 1996). In marine bivalves, positive heterosis in hybrids of different populations has been widely reported in scallops (Cruz and Ibarra, 1997; Zheng et al., 2006, 2011; Zhang et al., 2007; Wang and Li, 2010), oysters (Hedgecock et al., 1995; Hedgecock and Davis, 2007; Rawson and Feindel, 2012; Zhang et al., 2017a; Yin and

Table 4 Growth index of of two population progeny (ZZ and HH) and their reciprocal hybrids (ZH and HZ), at different days post-fertilization, as well as heterosis (H and I). Items

D larvae SL (μm)

ZZ ZH HZ HH H(%) IZH(%) IHZ(%)

151.16 154.90 154.43 149.60 2.85 2.47 3.23

± ± ± ±

Day7 SL (μm) 2.43a 4.50b 4.81b 3.06a

178.68 183.96 185.53 178.80 3.36 2.96 3.76

Day15 SL (μm) ± ± ± ±

3.14a 2.99a 2.50a 6.50a

212.73 226.00 263.27 242.33 7.52 6.24 8.64

Day 90 SL (mm) ± ± ± ±

8.39b 23.58b 8.48a 59.42a

3.18 ± 3.53 ± 3.96 ± 3.38 ± 14.18 11.01 17.16

Day 360 SL (mm) 0.43c 0.41b 0.26a 0.55b

38.84 45.06 51.28 43.65 16.79 16.01 17.48

± ± ± ±

Day360 WW (g) 3.99c 4.21b 4.56a 4.33b

10.96 14.87 19.29 13.35 40.52 35.68 44.49

± ± ± ±

1.15c 1.50b 1.83a 1.29b

H indicates mid-parent heterosis; and IZH and IHZ indicate the single parent heterosis of ZH and HZ groups, respectively. For growth traits (SL, WW), n = 90 (3 replicates × 30) in each experimental group. Different superscript letters in each column indicate significant differences (p < .05). 6

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Declaration of Competing Interest

Hedgecock, 2019) and abalone (You et al., 2015; Li et al., 2017, 2018) among others. If the populations crossed do not differ in their gene frequency, then there will be no heterosis. Conversely, heterosis will be greatest when one allele is fixed in one population and the other allele is fixed in the other population (Falconer and Mackay, 1996; Zhang et al., 2007). Our results show that there is a degree of phenotypic difference between the two populations of clams. Given this is likely due to genetic variation, heterosis is a reasonable explanation for the improved performance of the hybrid progeny. Furthermore, we used a two factors model to assess the effects of egg origin, mating strategy, and their interaction on growth and survival traits (Hedgecock and Davis, 2007). In terms of survival, rate of metamorphosis was predominantly affected by maternal origin, while mating strategy was the primary influence on other measures of survival. Growth traits were primarily affected by mating strategy, with the effects of maternal origin secondary, suggesting that breeding hybrids is a good way to improve growth performance. There were considerable differences between the performance of the two groups of reciprocal hybrids in this study as well as others bivalves (Zheng et al., 2006; Zhang et al., 2007, 2017a; Wang and Côté, 2012). According to our results, maternal origin had the greatest effect on the growth divergence and rate of metamorphosis of the hybrids, while the results of this study and those of previous studies on other marine bivalves suggest that mating strategy is important in determining production traits. In general, maternal origin caused the most variation in the early developmental stages (Falconer and Mackay, 1996; Zhang et al., 2007; Zheng et al., 2011). The effects of maternal inheritance on the growth traits of the reciprocal hybrids in this study persisted throughout the whole experiment, and single maternal-parent heterosis for growth traits was always positive for both ZH and HZ progeny.

There are no conflicts to all authors for this paper. Acknowledgments The authors would like to thank the editor and three reviewers for his assistance in the manuscript revision process. This work was supported by National Science Foundation of China (31872566), Strategic pilot project of the Chinese Academy of Sciences (XDA13020202; XDA13020403), Chinese Ministry of Science and Technology through the National Key Research and Development Program of China (2018YFC1406505, 2018YFD0901400), Guangzhou city programs, Guangdong province, China (201803020047; 201804020073), Open subject of key Laboratory for Aquatic Economic Animal Culture in the South China Sea (KFKT2019ZD10), Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences (ISEE2018PY01; ISEE2018ZD02; ISEE2018PY03), Open Foundation of State Key Laboratory of Loess and Quaternary Geology (SKLLQG1813), China Agricultural Shellfish Industry Technology System Project (CARS-49), and Science and Technology Planning Project of Guangdong Province, China (2017B030314052). References Alcazar, S.N., 1988. Spawning and larval rearing of Tridacnid clams in the Philippines. In: Copland, J.W., Lucas, J.S. (Eds.), Giant Clams in Asia and the Pacific. Australian Centre for International Agricultural Research, Canberra, pp. 125–128 Monograph No. 9. Benzie, J.A.H., 1993. Review of the population genetics of giant clams[C]//genetic aspects of conservation and cultivation of giant clams. ICLARM Conference Proceedings. 39, 1–6. Benzie, J.A.H., Williams, S.T., 1995. Gene flow among giant clam (Tridacna gigas) populations in Pacific does not parallel ocean circulation. Mar. Biol. 123, 781. Benzie, J.A.H., Williams, S.T., 1996. Limitations in the genetic variation of hatchery produced batches of the giant clam, Tridacna gigas. Aquaculture 139, 225–241. Benzie, J.A.H., Williams, S.T., 1997. Genetic structure of Giant clam (Tridacna maxima) populations in the West Pacific is not consistent with dispersal by present-day ocean currents. Evolution 51, 768–783. Benzie, J.A.H., Williams, S.T., 1998. Phylogenetic relationships among giant clam species (Mollusca: Tridacnidae) determined by protein electrophoresis. Mar. Biol. 132, 123–133. Braley, R.D., 1992. The Giant Clam: Hatchery and Nursery Culture Manual. Monograph. No. 15. Australian Centre of International Agricultural Research, Canberra. Braley, R.D., Militz, T.A., Southgate, P.C., 2018. Comparison of three hatchery culture methods for the giant clam Tridacna noae. Aquaculture 495, 881–887. Charlesworth, B., 2018. Mutational load, inbreeding depression and heterosis in subdivided populations. Mol. Ecol. 27, 4991–5003. Cruz, P., Ibarra, A.M., 1997. Larval growth and survival of two Catarina scallop (Argopecten circularis, Sowerby, 1835) populations and their reciprocal crosses. J. Exp. Mar. Bio. Ecol. 212, 95–110. Falconer, D.S., Mackay, T.F.C., 1996. Introduction to Quantitative Genetics (4th). Longman Scientific and Technical, London. Hedgecock, D., Davis, J.P., 2007. Heterosis for yield and crossbreeding of the Pacific oyster Crassostrea gigas. Aquaculture 272, S17–S29. Hedgecock, D., McGoldrick, D.J., Bayne, B.L., 1995. Hybrid vigor in Pacific oysters: an experimental approach using crosses among inbred lines. Aquaculture 137, 285–298. Hui, M., Kraemer, W.E., Seidel, C., Nuryanto, A., Joshi, A., Kochzius, M., 2016. Comparative genetic population structure of three endangered giant clams (Cardiidae: Tridacna species) throughout the indo-West Pacific: implications for divergence, coHHectivity and conservation. J. Molluscan Stud. 82, 403–414. Hui, M., Nuryanto, A., Kochzius, M., 2017. Concordance of microsatellite and mitochondrial DNA markers in detecting genetic population structure in the boring giant clam Tridacna crocea across the indo-Malay archipelago. Mar. Ecol. 38, e12389. Kochzius, M., Nuryanto, A., 2008. Strong genetic population structure in the boring giant clam, Tridacna crocea, across the indo-Malay archipelago: implications related to evolutionary processes and coHHectivity. Mol. Ecol. 17, 3775–3787. Kong, L., Song, S., Li, Q., 2017. The effect of interstrain hybridization on the production performance in the Pacific oyster Crassostrea gigas. Aquaculture 472, 44–49. Li, J., Wang, M., Fang, J., Liu, X., Mao, Y., Liu, G., Bian, D., 2017. Reproductive performance of one-year-old Pacific abalone (Haliotis discus haHHai) and its crossbreeding effect on offspring growth and survival. Aquaculture 473, 110–114. Li, J., Wang, M., Fang, J., Liu, X., Xue, S., Mao, Y., Liu, G., 2018. A comparison of offspring growth and survival among a wild and a selected strain of the Pacific abalone (Haliotis discus hannai) and their hybrids. Aquaculture 495, 721–725. Lucas, J.S., 1994. The biology, exploitation, and mariculture of giant clams (Tridacnidae). Rev. Fish. Sci. 2, 181–223.

4.3. Practical applications In this study, the performance of the HH progeny in terms of survival and growth was superior to that of the ZZ progeny under artificial hatchery conditions, suggesting that the difference could be caused by physiological adaptation Our results thus provide evidence for the selection of this strain for aquaculture and a basis for selective breeding based on phenotype. Heterosis was found in adults as well as larval stages, and it increased as growth increased. The growth and survival rates of the two reciprocal cross groups were greater than those of the two pure populations. HZ progeny in particular showed tremendous heterosis compared with the two pure populations. Therefore, the results of this study suggest that the hybrid HZ strain would be an excellent choice for the giant clam aquarium market and aquaculture industry in the South China Sea. 5. Conclusions Due to the long-term geographic isolation of the two populations, there are considerable phenotypic differences between their progeny. The Huangyan Island population's progeny were larger and faster growing with a higher rate of metamorphosis and the selection of this population for aquaculture is therefore potentially advantageous. Crossbreeding between the two populations produced considerable improvements in performance compared to the pure populations and there did not appear to be any fertilization barrier. HZ progeny exhibited particularly fast growth and a high rate of metamorphosis compared to the other three groups, and could in future be used as an improved genetic line in boring giant clam aquaculture. Author statement Giant clam parents were collected with the approval of the Hainan provincial government (China) and with minimal impacts on the local ecological environment. Ethics approval was not required for this study. 7

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