Performance evaluation of reciprocal hybrids derived from the two brackish oysters, Crassostrea hongkongensis and Crassostrea sikamea in southern China

Aquaculture 473 (2017) 310–316

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Performance evaluation of reciprocal hybrids derived from the two brackish oysters, Crassostrea hongkongensis and Crassostrea sikamea in southern China Yuehuan Zhang 1, Jun Li 1, Yang Zhang, Haitao Ma, Shu Xiao, Zhiming Xiang, Ziniu Yu ⁎ 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 Sciences, Guangzhou 510301, China South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510275, China

a r t i c l e

i n f o

Article history: Received 13 August 2016 Received in revised form 26 January 2017 Accepted 24 February 2017 Available online 27 February 2017 Keywords: Crassostrea hongkongensis Crassostrea sikamea Interspecific hybridization Phenotypic traits Heterosis

a b s t r a c t Interspecific hybridization has been used as an important tool for genetic manipulation in oyster culture with the aim to produce a new strains having desired traits from both the parental species. A two by two factorial cross between sympatric oyster species, Hong Kong oyster (Crassostrea hongkongensis) and Kumamoto oyster (Crassostrea sikamea), yielded hybrids that were fast growing (a characteristic of Hong Kong oyster) and had superior meat quality (characteristic to Kumamoto oyster). Reciprocal hybrids were obtained by the acceptable level of fertilization and hatching rate compared to these of two parental species. The survival rate of the hybrids was similar to that of the parental species. The hybrids exhibited fully developed gonads and gametes when sexually mature. Relative to C. sikamea, the hybrids also showed significant growth advantages as in shell height and wet weight. The shell morphology of the hybrid was dictated by maternal inheritance. Our results revealed that reciprocal hybrids exhibited larger growth heterosis and survival advantage (in terms of C. sikamea), as well as a higher meat weight, showing great potential for application in the oyster aquaculture industry in southern China. Statement of relevance: We firstly conducted the interspecific hybridization between the largest (Crassostrea hongkongensis) and smallest oysters (C. sikamea) in Crassostea genus, and then obtained reciprocal hybrids with obvious the heterosis (in terms of C. sikamea). These hybrids are very useful as a new stock for the oyster aquaculture industry. © 2017 Elsevier B.V. All rights reserved.

1. Introduction In China, oysters are important species in shellfish industry accounting for N30% of the total marine mollusca yield with a total production of 4.4 million tons in 2014 (People's Republic of China Ministry of Agriculture, 2015). Five commercially important oysters that are commonly found along the coast of China belong to the genus Crassostrea. Specifically, C. gigas (Thunberg, 1793), C. angulata (Lamarck, 1819), C. ariakensis (Fujita, 1913), C. hongkongensis (Lam & Morton, 2003) and C. sikamea (Amemiya, 1928) have been widely cultivated for their commercial value (Wang et al., 2008a). Depending upon their environmental adaptability, they were divided into two major types: the saltwater oysters (C. gigas and C. angulata) and the brackish oysters (C.

⁎ Corresponding author at: South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China. E-mail address: [email protected] (Z. Yu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.aquaculture.2017.02.031 0044-8486/© 2017 Elsevier B.V. All rights reserved.

hongkongensis, C. arikensis, C. sikamea) (Wang et al., 2004; Wang et al., 2008b; Li et al., 2013). The Hong Kong oyster Crassostrea hongkongensis and the Kumamoto oyster C. sikamea are two brackish oysters in southern China, which inhabit in the same geographical area (Wang and Guo, 2008a). The Hong Kong oyster is one of the most important endemic and cultured oyster species. It has a high market value, a cultivation history of nearly one thousand years and an annual production of 1.6 million metric tons along the south China coast (Huo et al., 2014; Zhang et al., 2016; People's Republic of China Ministry of Agriculture, 2015), Its distribution ranges from Fujian to Guangxi provinces, with a population center at Guangdong province (Guo et al., 2006; Guo, 2009). Since 2006, several hatcheries have begun to produce the Hong Kong oyster grown under artificial conditions; however most of using seeds still derived from natural collections. The Kumamoto oyster not only occurs naturally in China, but also lives in abundance over a wide geographical distribution, ranging from Jiangsu to Guangxi, including Hainan Island, with population centered in Zhejiang province. Traditionally, Kumamoto oyster is considered as an important wild fishery resource; mostly collected by

Y. Zhang et al. / Aquaculture 473 (2017) 310–316

farmers from the reefs and stones in the inter-tidal zone (Wang et al., 2013). The Hong Kong oyster has several desirable qualities of commercial importance, such as fast growth, large size and long life span. Aiming towards oyster genetic improvement, these are traits one would like to retain in hybrid progeny. For example, the fast growth trait of C. hongkongensis would be a highly desirable characteristic for the slow growing cultivated variety C. sikamea. Furthermore, successful transfer of the trait for fast growth of C. hongkongensis to C. sikamea could potentially shorten its culture cycle and reduce farming risks. On the other hand, high meat quality of the Kumamoto oyster can be desired trait for Hong Kong oyster itself. Thus, hybridization between C. hongkongensis and C. sikamea could be potentially beneficial for the generation of a hybrid variety with improved genetic traits. Interspecific hybridization has been reported to increase growth, improve meat quality, and strengthen disease resistance of hybrids (Hulata, 1995; Hu et al., 2012). Such hybridization not only produces heterosis but is also an effective method to expand species variation and breed new varieties (Bartley et al., 2001). However, not all interspecific hybridization results in heterosis. For example, when a hybrid inherits traits from its parents that are not fully compatible, fitness can be reduced leading to what is known as out breeding depression or hybrid breakdown (Stelkens and Seehausen, 2009). Previously, several hybridizations with molecular confirmation among the genus Crassostrea have been attempted in China (Table 1). Although the hybrids could survive to adulthood, the growth divergence of hybrids was observed in different crosses (Table 2). So far, there have been no reports of hybridization between the two brackish oysters, C. hongkongensis and C. sikamea. This may be because C. hongkongensis is a newly identified species that had been wrongly classified as C. rivularis owing to the similar appearance of the two species (Boudry et al., 2003; Lam and Morton, 2003; Wang et al., 2004; Wang and Guo, 2008a). To obtain a new potential oyster stock having the combination of rapid growth characteristics of C. hongkongensis and the high meat quality trait of C. sikamea, a series of 2 × 2 factorial cross experiments were conducted between the two species. The phenotypic characters, including fertilization, survival, growth, and gonad development of C. hongkongensis progeny, C. sikamea progeny, and their reciprocal hybrids were compared under conditions that mimicked the coast of southern China. Heterosis of the hybrids was analyzed throughout the entire life cycle. The identity of the parental species and their hybrids were confirmed by molecular markers. 2. Materials and methods 2.1. Brood stock animal collection and strip spawning Sexually mature two-year-old Crassostrea hongkongensis from Zhuhai and one-year-old C. sikamea from Zhanjiang, the Guangdong

311

Table 2 Fertilization success (√) and failure (×) among five important oysters in China, H = C. hongkongensis, R = C. ariakensis, G = C. gigas, A = C. angulata, S = C. sikamea. Parents

H♂

R♂

G♂

A♂

S♂

H♀ R♀ G♀ A♀ S♀

HH × × × √

√ RR √ √ √

√ √ GG √ √

√ √ √ AA √

√ × × × SS

Note: Hybridization between C. gigas and C. angulata is not a true interspecific cross, because these two oysters have been defined as two subspecies under one species.

province were collected, and transported to the Zhanjiang Marine Economic Animal Station hatchery at the South China Sea Institute of Oceanology of the Chinese Academy of Sciences in early May 2014. These two species were then maintained under controlled artificial conditions till adulthood. They were identified based on their shell morphology and gill tube structure as described by Wang et al. (2004). All brood stock animals were reared for one week in indoor conditions with temperatures from 26.3 to 27.5 °C and salinity from 18 to 20 ppt. The gonad tissue of each animal was individually dissected under a light microscope (Olympus) to determine its sex. Thirty healthy females and thirty healthy males from each species were selected for the experiments. The sperms and eggs of each individual were removed from mature gonads and were processed following specific protocol and then suspended in sand-filtered seawater in individual beakers. Eggs from each female were rinsed with sand-filtered seawater, followed by a 90-μm nylon screen to remove detritus, collected with a 25-μm nylon screen and re-suspended in seawater in individual beakers. Sperm from each selected male were collected in separate beakers and diluted into one beaker to achieve the same density before pooling according to Xu et al., 2009. The beakers were maintained at temperatures 26.8 to 27.9 °C and salinity 18 to 20 ppt. After the gametes were collected, tissues of the spawned animals were fixed using 95% ethanol for subsequent confirmation with genetic markers as described by Wang and Guo (2008a). 2.2. Cross-fertilization For each species, eggs from three females were pooled and then divided equally into two 5-L beakers. Prior to fertilization eggs were checked to see if any uncontrolled fertilization has occurred. Absence of any polar bodies made the eggs fit for interspecific hybridization. Each beaker of eggs was fertilized with a mixture of sperm from three C. hongkongensis (H) and three C. sikamea (S), with a ratio of 20–25 sperm surrounding an egg. Thus, artificial crosses were performed with the following four different combinations: C. hongkongensis

Table 1 Interspecific hybridization among Crassostrea oysters in China. Hybridized crosses

Main results and comments

Reference

C. hongkongensis × C. gigas

One way fertilization, slow growth and a high degree of sterility.

C. hongkongensis × C. angulata C. hongkongensis × C. ariakensis C. hongkongensis × C. sikamea C. gigas × C. angulata

One way fertilization, fast growth and complete fertility.

Zhang et al. (2012a, 2014, 2015a, 2015b, 2016) Zhang et al. (2015a)

One way fertilization, fast growth (Compared with C. hongkongensis) and complete fertility

Huo et al. (2013, 2014)

Two way fertilization, fast growth (Compared with C. sikamea), improve meat quality, and complete fertility Two way fertilization, moderate growth, improve high temperature resistance, and complete fertility Two way fertilization, hardly any growth heterosis and complete fertility. One way fertilization, fast growth (Compared with C. sikamea) and complete fertility. Two way fertilization, slow growth and complete fertility. One way fertilization, slow growth and complete fertility. Two way fertilization, fast growth (Compared with C. sikamea) and complete fertility.

In this study

C. gigas × C. ariakensis C. gigas × C. sikamea C. ariakensis × C. augulata C. ariakensis × C. sikamea C. angulata × C. sikamea

All oyster interspecific hybrids showed survival till adulthood, with environmental factors playing a key role in the survival rate.

Zheng et al. (2012) Zhang et al. (2012b) Su (2015) Yao et al. (2015) Xu et al. (2009, 2011) Xu et al. (2014)

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♀ × C. hongkongensis ♂ (HH), C. hongkongensis ♀ × C. sikamea ♂ (HS), C. sikamea ♀ × C. hongkongensis ♂ (SH), and C. sikamea ♀ × C. sikamea ♂ (SS). For interspecific hybrids, approximately 50% more sperm were added to increase the chances of successful fertilization. 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 60-L bucket at a density of 30–40 eggs/mL for hatching. Zygotes developed in sand-filtered seawater maintained at temperatures 26.7 to 27.5 °C and salinity of 20 ppt. The whole experiment was repeated 3 times using 3 sets of parents, with each set constituting three pooled females and three pooled males (Table 3). 2.3. Larval rearing and spat grow-out Twenty-four hours after fertilization, D-larvae from each cross, HH, HS, SH and SS, were collected on a 40-μm sieve and reared separately in a 500-L bucket. The initial larval density was adjusted to 5 larvae/ mL, and was maintained at this level by controlling the water volume. Seawater was completely changed once every three days. The larvae were fed on Isochrysis galbana for the first 6 days and mixture of Platymonas subcordiformis and I. galbana (the volume ratio = 1:1) after day 6, with density gradually increasing from 6000 to 80,000 cells/mL/day. The rearing water was maintained at 27.0–29.2 °C with a salinity of 18–20 ppt. When 50% of the larvae developed eyespots and feet (after the 16– 20 d larval rearing stage), strings of corrugated plastic plates were placed in buckets as cultch to induce larval settlement. Larvae set within 7 days, and newly settled spat were nursed in buckets for another two weeks to prevent contamination from wild spat. Subsequently, all spat were transported to concrete tanks (5.0 × 4.0 × 1.2 m3) and fed with Chlorella vulgaris at 80,000–10,000 cells/mL/day. 30% of the water in the tanks was changes once a day. At day 60, spat were detached, transferred into spat bags and hung on suspended long lines in Beibu Bay. Spat bags were changed from small to larger mesh sizes every 2 weeks. During the grow-out period from July 2014 to June 2015, water temperatures ranged from 15.2 to 30.6 °C, and salinity ranged from 12 to 27 ppt. 2.4. Genetic confirmation For every experimental group, 90 individuals (3 replicates × 30) were examined at day 360. DNA was extracted from ethanol-fixed samples from the reciprocal hybrids and two intraspecific groups using the TIANamp Marine Animals DNA kit (Tiangen). Genetic confirmation of reciprocal hybrids were conducted using the ITS2 (internal transcribed spacer 2) marker (Wang and Guo, 2008b). Primer sequences used for ITS2 are 5′-TCTCGCCTGATCTGAGGTCG-3′ (5.8S forward) and 5′GCAGGACACATTGAACATCG-3′ (18S reverse). PCR was performed in

Table 3 Experimental design for the hybridization between C. hongkongensis and C. sikamea. Parents

H1♂

S1♂

H2♂

S2♂

H3♂

S3♂

H1♀ S1♀ H2♀ S2♀ H3♀ S3♀

HH1 SH1 – – – –

HS1 SS1 – – – –

– – HH2 SH2 – –

– – HS2 SS2 – –

– – – – HH3 SH3

– – – – HS3 SS3

HH and SS indicate the intraspecific crosses C. hongkongensis ♀ × C. hongkongesis ♂, and C. sikamea ♀ × C. sikamea ♂, respectively; HS indicates the interspecific cross C. hongkongensis ♀ × C. sikamea ♂, SH indicates the interspecific cross C. sikamea ♀ × C. hongkongensis ♂. The subscript number 1, 2, 3 denotes three replicates, and each replicate was conducted by pooled pair mating.

25-μL volumes containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 μM of each primer, 20 ng of template DNA, 1 U Taq polymerase, 2.5 μL of 10 × PCR buffer, and 0.4 mg/mL BSA. The thermal cycler protocol consisted of an initial denaturation at 95 °C for 5 min, 30 cycles at 95 °C for 1 min, 62.5 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. Four controls were included in the experiment: DNA from an identified C. hongkongensis parent, DNA from a C. sikamea parent, and two others using DNA from the reciprocal hybrids. For species identification based on ITS2 fragment length, all amplified fragments were separated on 2% agarose gels containing 0.2 μg/mL ethidium bromide, and visualized under a UV transilluminator. A set of species-specific COI primers was further utilized to identify the maternal heredity of offspring (Wang and Guo, 2008a). The universal PCR primers used for mitochondria COI are 5′-GGTCAACAA ATCATAAAGATATTGG-3′(LCO1490) and 5′-TAAACTTCAGGGTGACC AAAAAATCA-3′ (HCO-2198). Species-specific primers used for this particular gene are 5′-GGAGTAAGTGGATAAGGGTGGATAG-3′ for C. hongkongensis with a product length of 387-bp, and 5′ AAGTAACCTTA ATAGATCAGGGAAC(A)C-3′ for C. sikamea with a product length of 546-bp. The COI fragments were amplified with an initial denaturation at 95 °C for 2 min, 30 cycles at 95 °C for 1 min, 51 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. Three controls (DNA from an identified C. hongkongensis parent, DNA from a C. sikamea parent, and DNA from reciprocal hybrid) were also included in this experiment. Amplified fragments were separated on 2% agarose gels, containing 0.2 μg/mL ethidium bromide, and visualized under a UV transilluminator. Female parents of hybrids were identified by the specific length of their PCR products. 2.5. Sampling and measurements The embryo development index (egg diameter, cleaved rate and D larvae rate), cumulative survival rate (at day 15, day 90 and day 360), growth parameter (D larvae size at day 15, day 90 and day 360) of each group were determined as previously reported (Zhang et al., 2014). Cumulative survival rate was defined as the ratio between the numbers of individuals at different developmental stages to that of D larval stage. Specifically, 3600 spats (approximately 10 mm in shell height) of each group were put into 12 bags with 5 mm mesh at day 60. The spat bags were changed monthly with bags of increasing mesh size ranging from 5 mm to 20 mm. As the spat grew, the density was randomly adjusted monthly from 300 ind./bag to 50 ind./bag. Moreover, the dead oysters were discarded at every bag change, and the density of each group was readjusted to maintain similar levels among various groups. Differences in hatching index, survival rate and growth parameter between groups and replicates were analyzed using multiple comparisons of means using a two-way ANOVA. To improve the normality and homoscedasticity, the hatching rate and survival rate were arcsinetransformed prior to analysis, and growth parameters were transformed to a logarithmic scale with base 10 of base. All statistical analyses were performed using SPSS 18.0. To access the significance, the cutoff p b 0.05 was set as the criterion for all experiments, unless noted otherwise. To evaluate the aquaculture traits, heterosis was used in this study. Mid-parental heterosis (H) was used (Zhang et al., 2007, 2014; Wang et al., 2011) and calculated using the following equation: Hð%Þ ¼ ½X F1 −ðXH þ XS Þ=2  100=ðXH þ XS Þ where XF1 indicates the mean phenotypic value (shell height, survival rate, etc.) of reciprocal hybrids, while XH and XS indicates the mean phenotypic value (shell height, survival rate, etc.) on the same day for the Hong Kong oyster and the Kumamoto oyster, respectively. To determine the effects of the egg origin and mating strategy (intravs. interspecific crosses) on the survival and growth, a two-factor

Y. Zhang et al. / Aquaculture 473 (2017) 310–316

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analysis of variance was used (Cruz and Ibarra, 1997; Zhang et al., 2007), as follows: Yijk ¼ μ þ EOi þ MS j þ ðEO  MSÞij þ eijk Here, Yijk is the mean shell height (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 height (wet weight or survival rate) (i = 1, 2). MSj is the mating strategy effect on the shell height (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). To further estimate the increase in production traits of the hybrids compared with that of the Kumamoto oyster, the production increase (I) was calculated by the formula (Wang et al., 2011; You et al., 2015):

Fig. 1. Agarose gels (2%) showing the amplified PCR products of the complex COI (above) and ITS2 (below) of C. hongkongensis, C. sikamea, and their hybrids. Note: M, marker DL1000; 1–2, C. hongkongensis parent; 3–5, hybrid spats from C. hongkongensis female × C. sikamea male cross; 6–8, hybrid spats from C. sikamea female × C. hongkongensis male cross; 9–10, C. sikamea parent.

Ið%Þ ¼ ðX F1 −XAi Þ  100=XAi where XF1 is the phenotypic value (shell height, survival rate, etc.) of the reciprocal hybrid progeny, and XAi in the mean phenotypic value (shell height, survival rate, etc.) of Kumamoto oyster.

3.3. Cultured traits

3. Results

3.3.1. Survival During the larvae stage, the survival rate of all larvae gradually decreased as the larvae grew older. At day 15, the survival rate of all the four experimental groups exceeded 80%, with no significant difference among groups (p N 0.05). After metamorphosis, all spat were transferred to the grow-out environments. The survival rates of reciprocal hybrid spat were significantly higher than the two intraspecific crosses from day 90 to 360 (p b 0.05). Specifically, at day 360, the survival rate was 36.4% in HH, 48.0% in SS, 52.5% in HS, and 53.3% in SH, with mid-parental heterosis of 25.3; whereas the survival advantage of IHS and ISH was 9.3 and 11.0, respectively (Tables 4, 5).

3.1. Hatchery A notable difference between C. hongkongensis and C. sikamea is the size of their eggs. The eggs of C. hongkongensis (49.7 μm) were significantly larger than those of C. sikamea (43.7 μm) (P b 0.001). The fertilization rate of reciprocal hybrids were significantly lower than those of the two parental species progeny (p b 0.05), suggesting that there existed a certain degree of the sperm-egg recognition barriers between the two brackish oysters. The D larvae stage of the reciprocal hybrids was prolonged from 2 h to 3 h compared to the two intraspecific crosses. Survival of fertilized eggs to D-stage larvae was 48.4% for HS, and 77.7% for SH; both of which were significantly lower than HH (89.0%) and SS (86.1%) (Table 4).

3.3.2. Growth As for the D larvae, a significant difference was noted in shell height among the four groups. Reciprocal hybrid larvae were similar to that of maternal parent progeny, with a mid-parental heterosis of 2.1% (Tables 5, 6). At day 15, shell height of eyed larvae of the reciprocal hybrid was slightly larger than that of two parental progeny, however without any statistical significance (p N 0.05). At day 90, the size of SH larvae was median to that of the two parental spats and were significantly different from both (p b 0.05), while shell height of HS spat was found to be similar to those of HH, but was significantly larger than the other two groups (p b 0.05). At day 360, the growth trend of all the progeny was similar to the day 90. The mid-parental heterosis at day 360 was 13.4% for shell height and 19.6% for wet weight. Notably, compared with those of C. sikamea, IHS was 99.8% for shell height and 299.7% for wet weight; ISH was 55.9% for shell height and 166.3% for wet weight, at day 360 (Tables 5, 6).

3.2. Genetic confirmation A single specific COI band was produced in both C. hongkogensis and C. sikamea with approximate band lengths of 387 bp and 546 bp, respectively. Furthermore, the HS progeny produced a 387 bp band as that of C. hongkongensis, while the SH progeny produced bands similar to C. sikamea of 546 bp, indicating that matrilineal inheritance of mitochondrial genes occurs during oyster hybridization. C. hongkongensis and C. sikamea progeny produced single bands with ITS2 at approximately 600 bp and 650 bp, respectively. All hybrid progeny produced two bands: one at approximately 600 bp and the other at approximately 650 bp, indicating that the reciprocal hybrids inherited nuclear DNA from both parents and that they are true hybrids (Fig.1).

Table 4 Hatching index, cumulative survival rate of C. hongkongensis (HH), C. sikamea (SS), and their hybrids (HS and SH), and heterosis (H and I). Items

HH SS HS SH H (%) IHS (%) ISH (%)

Hatching index

Cumulative survival rate (%)

Egg-diameter (μm)

Cleaved rate (%)

49.7 ± 0.9a 43.7 ± 1.1b – – – – –

95.2 92.1 57.2 82.1 – – –

± ± ± ±

1.2a 3.1a 4.3c 7.8b

D-stage (%) 89.0 86.1 48.4 77.7 – – –

± ± ± ±

2.2a 1.5a 6.1c 6.7b

Day 15 82.9 83.9 86.0 87.1 3.8 2.5 3.9

± ± ± ±

Day 90 5.2a 6.8a 6.3a 5.1a

57.2 64.8 68.5 69.4 13.1 5.7 7.1

± ± ± ±

Day 360 5.7b 6.8a 4.1a 4.7a

36.4 48.0 52.5 53.3 25.3 9.3 11.0

± ± ± ±

4.5b 5.6a 5.3a 6.4a

H indicates mid-parent heterosis; and IHS and ISH indicate the single parent heterosis of HS and SH groups, respectively. For cleavage rate, D-stage rate, cumulative survival on different days, n = 9 (3 replicates × 3), and for egg-diameter, n = 90 (3 replicates × 30 individuals) in each experimental group. Different superscript letters in each column indicate significant difference (p N 0.05).

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Table 5 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

Source

Day 15

Day 90

Day 360

df

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

1 1 1 1 1 1 1 1 1

Survival rate

Shell height

MS

F-value

MS

F-value

b0.001 0.003 0.054 0.026 0.010 0.004 0.026 0.010 0.004

0.925 0.212 b0.001⁎⁎⁎ 0.048⁎

0.001 b0.001 b0.001 0.046 0.019 b0.001 0.595 1.521 b0.001

b0.001⁎⁎⁎ b0.001⁎⁎⁎ 0.473 0.001⁎⁎ 0.038⁎

0.206 0.434 0.048⁎ 0.206 0.434

0.814 b0.001⁎⁎⁎ b0.001⁎⁎⁎ 0.827

3.3.3. Gonad development To assess the reproductive potential of reciprocal hybrids, we examined gonadal samples during the reproductive stage. The gonads of oysters from four groups were fully mature and had either functional eggs or sperm. The sex ratio was normal and no significant difference was observed on Chi-Square Tests. The percentage of females were 67.3% in HS, 60.9% in SH, 71.6% in HH, and 65.4% in SS, while male percentage were 33.7% in HS, 39.1% in SH, 28.4% in HH, and 34.6% in SS (Table 7). 3.4. Morphological traits Abundant cuticular tissue was present in the right valve of the HS progeny, a morphological feature similar to C. hongkongensis. SH progeny, on the other hand, lacked the cuticle, a phenotype similar to the other parent C. sikamea (Fig.2). In the left shell, number of ribs from ranged from 7 to 9 in the SH progeny, similar to SS parental type, while no ribs were observed on the left valve of HS, a phenotype similar to SS (Table 8, Fig. 2). These morphological evidences suggest that shell form of reciprocal hybrids followed maternal genetics. The value of SW/SH, SL/SH and SW/SL was significantly different between two parental species, while these of reciprocal hybrids lay to two parents and tend to their maternal progeny, respectively (Table 8). To sum up, the elongated shell morphology of HS progeny was similar to those of C. hongkongensis, while the round or oval-shaped of SH progeny resembled C. sikamea, exhibiting remarkable maternal effects. 4. Discussion Hybridization, as a means of species modification, plays a very important role in the speciation and adaptive radiation of animals (Hulata, 1995). Moreover, this breeding technique is used by aqua culturists in the hope of producing aquatic organisms with specific desirable traits or general improvement in performance. Commonly, the desired goal is to produce offspring that perform better than both or Table 6 Shell size (SL and SH) and whole wet weight (WW) of C. hongkongensis (HH), C. sikamea (SS), and their hybrids (HS and SH) at different days post-fertilization, as well as heterosis (H and I). Items

D larvae SL (μm)

HH SS HS SH H IHS ISH

70.4 64.3 70.7 66.8 2.1 10.0 3.9

± ± ± ±

1.0a 1.1b 0.9a 1.0ab

Day15 SH (μm) 307.3 296.5 328.4 309.8 5.7 10.8 4.5

± ± ± ±

Day 90 SH (mm) 27.2a 25.7a 23.5a 24.6a

54.8 31.0 51.3 42.4 9.3 65.8 36.9

± ± ± ±

6.7a 3.2c 4.9a 4.3b

Day 360 SH (mm) 96.8 45.3 90.4 70.6 13.4 99.8 55.9

± ± ± ±

10.5a 5.9c 9.3a 7.5b

Day360 WW (g) 86.4 ± 18.9 ± 75.6 ± 50.4 ± 19.6 299.7 166.3

12.7a 3.4c 9.8a 7.2b

H indicates mid-parent heterosis; and IHS and ISH indicate the single parent heterosis of HS and SH groups, respectively. For growth trait (SH, WW), n = 90 in each experimental group. Different superscript letters in each column indicate significant difference (p b 0.05).

Table 7 Number and percent of females and males in one-year-old C. hongkongensis (HH), Crassostrea sikamea (SS), and their reciprocal hybrids (HS and SH). Items

Female

Male

Total

HH SS HS SH

54 (67.5%) 61 (76.3%) 67 (83.8%) 61 (76.3%)

26 (32.5%) 19 (23.7%) 13 (16.2%) 19 (23.7%)

80 (100%) 80 (100%) 80 (100%) 80 (100%)

single parental species (Barton, 2001). This study clearly demonstrates that, two way hybridization between two sympatric species of oyster having different economically viable characteristic yield superior progeny than the parental species. Furthermore, the hybrids exhibited larger heterosis (in terms of C. sikamea) in growth as well as some features that may be immediately applicable to the aquaculture industry. 4.1. Fertilization Fertilization rate is a key parameter utilized to assess for commercial production of interspecies hybrids (You et al., 2015). Our study clearly demonstrates that hybridization between C. hongkongensis and C. sikamea is achievable using either one of them as sperm donor (twoway fertilization). However, the fertilization rates of reciprocal hybrids were lower than both the parental species, and exhibited asymmetry in fertilization strength. Even with some disadvantages, the fertilization rate of HS (82.1%) and SH (57.2%) was acceptable for large scale production mainly due to the abundant fecundity of eggs of the mature brood stocks for. Previous reports suggest that the fertilization difference that commonly appear on interspecific hybridization among Crassostrea genus, was mainly caused by differentiation of gamete recognition proteins (GRPs) between sperms and eggs (Wu et al., 2011). Among the five Crassostea oyster species (C. gigas, C. angulata, C. sikamea, C. hongkongensis, C. ariakensis), successful interspecific hybridization could only be achieved between C. hongkongensis and C. sikamea, as demonstrated in this study (Table 2). However, eggs from C. hongkongensis and C. sikamea can identify sperms from all other four oyster species, implying asynchronous evolution of sperm and eggs under brackish environments (Banks et al., 1994; Springer et al., 2008; Zhang et al., 2012a). Because fertilization rate can be influenced by water temperature, salinity, concentration of sperm, and gamete age, it is necessary to investigate the factors influencing fertilization rate between these two species in the future. These studies would help increase and stabilize the fertilization rates for future aquaculture applications. 4.2. Survival Reports from various crossbreeding experiments among Crassostrea genus suggested lower survivability or un-viability of interspecific hybrids, particularly during the early development stages (Allen et al., 1993; Gaffney and Allen, 1993; Xu et al., 2009, 2014; Zhang et al., 2012a; Batista et al., 2007, 2008). On the contrary, our results showed higher survival of hybrids with positive advantages throughout its whole life time. This could result from the better adaptability of reciprocal hybrids than that of two intraspecific cross progenies in particular environmental conditions (Johnson and Wade, 1996; Zhang et al., 2014, 2015a). Viability of the aquaculture animal is a very important performance trait, which is closely related to production. The viability of an aquaculture animal is known to be affected by the environment (Dégremont et al., 2010; Rawson and Feindel, 2012). As in the review paper by Gaffney and Allen (1993), a larger number of interspecific hybrids among Crassostrea genus are unviable. Nonetheless, we believed that if hybrid larvae can be produced, they could be settled, until adulthood following established hybridization protocol (Table 1). Here, it is

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Fig. 2. The cuticle surface of right shell (Right valves) and the number of ribs of left shell (Left valves) for C. hongkongensis, C. sikamea and their hybrids. Note: HH, C. hongkongensis progeny; HS, C. hongkongensis ♀ × C. sikaema ♂ progeny; SH, C. sikaema ♀ × C. hongkongensis ♂ progeny; SS, C. sikaema progeny.

observed that interspecific hybrids among Crassostrea genus are viable, but the survivability is dependent on both the genetic make up of the species and the environment in which it is cultured.

4.3. Growth Among the five Crassostrea species, the Hong Kong oyster C. hongkongensis and the Kumamoto oyster C. sikamea show maximal growth divergence (Lam and Morton, 2003; Wang et al., 2004; Guo et al., 2006; Guo, 2009). Usually, the Hong Kong oyster can grow up to 120–150 mm (shell height), 200–300 g (wet weight); while those of the Kumamoto oyster can grow up to 50–70 mm (shell height), 25– 50 g (wet weight) by two years (Li et al., 2013). The largest wild Hong Kong oyster, about 8–10 years in age, was found to have a shell height of 420 mm and wet weight of 1800 g (Lam and Morton, 2003; Wang et al., 2004); whereas the largest wild Kumamoto oyster, about 3 years in age, was 75 mm in shell height, and 63 g in wet weight (Data not shown). In this study, the Hong Kong oyster grew to 96.8 mm in shell height, 86.4 g in wet weight; while the Kumamoto oyster grew to 45.3 mm in shell height, and 18.9 g in wet weight at day 360. The size of HS hybrids was significantly larger than that of SH progeny; and both of them were in turn significantly larger than SS. These observations indicate that the growth trait of C. hongkongensis was partly transferred to reciprocal hybrids showing remarkable maternal effects. Our results suggested that hybridization could be used as a potent method to improve growth traits.

Table 8 Measure and countable traits of C. hongkongensis (HH), Crassostrea sikamea (SS), and their reciprocal hybrids (HS and SH) in one-year-old. Items

SW/SH

HH HS SH SS

0.35 0.37 0.44 0.47

± ± ± ±

SL/SH 0.04a 0.05a 0.05b 0.06b

0.57 0.59 0.68 0.71

± ± ± ±

SL/SW 0.03a 0.04a 0.07b 0.08b

0.63 0.60 0.68 0.66

± ± ± ±

Number of ribs 0.05a 0.06a 0.08a 0.09a

0 0 7–9 7–9

In case of oyster hybridization, growth heterosis and inferior of hybrids has been contradictorily reported in the past. Growth heterosis of hybrids was found on the crosses of C. hongkongensis ♀ × C. angulata ♂ (Zhang et al., 2015a), C. hongkongensis ♀ × C. ariakensis ♂ (Huo et al., 2013, 2014), C. sikamea ♀ × C. gigas ♂ (Su, 2015) and C. sikamea ♀ × C. angulata ♂ (Xu et al., 2014), while others yielded inferior hybrids (Gaffney and Allen, 1993; Allen and Gaffney, 1993; Allen et al., 1993; Huvet et al., 2004; Batista et al., 2007; Xu et al., 2009; Zhang et al., 2012a, 2015a). Two reasons may explain the growth difference in oyster hybrids. First, the genetic differences of between carious species produce the growth variation of hybrids (Sheridan, 1997; Lippman and Zamir, 2007; Hu et al., 2013). Second, the different cultured environmental conditions may contribute to the phenotypic difference of hybrids (Evans and Langdon, 2006; Swan et al., 2007; Rawson and Feindel, 2012; Proestou et al., 2016). 4.4. Shell traits Shell morphology and number of ribs in reciprocal hybrids were similar to that of their female parental species, indicating strong maternal effects. This phenomenon was similar to hybridization between the Japanese Patinopecten yessoensis and the Chinese scallop Chamys farreri which often resulted in progeny that resemble only one parent (Wang et al., 2011). In fact, all oyster hybrids exhibited maternal genetics as evidenced from shell morphology, cuticle surface, number of ribs, etc., indicating that shell traits was mostly determined by the female species during the hybridization process. However, the hybridization between Argopecten purpuratus and Argopecten irradians irradians exhibited contribution from two parental genes for shell traits (Wang et al., 2011; Hu et al., 2013). 4.5. Applicant prospects Reciprocal hybrids with faster growth (to C. sikamea) and high meat quality were obtained by two way oyster hybridization, and they were fully adapted to brackish environments in southern China. The size of HS progeny was slightly smaller but with a superior meat quality than that of C. hongkongensis. On the other hand SH progeny was remarkably

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