Selection for growth performance of tank-reared Pacific white shrimp, Litopenaeus vannamei

Seven growth-related traits were measured to assess the selection response and genetic parameters of the growth of Pacific white shrimp, Litopenaeus vannamei, which had been domesticated in tanks for more than four generations. Phenotypic and genetic parameters were evaluated and fitted to an animal model. Realized response was measured from the difference between the mean growth rates of selected and control families. Realized heritability was determined from the ratio of the selection responses and selection differentials. The animal model heritability estimate over generations was 0.44±0.09 for body weight (BW), and ranged from 0.21±0.08 to 0.37±0.06 for size traits. Genetic correlations of phenotypic traits were more variable (0.51–0.97), although correlations among various traits were high (>0.83). Across generations, BW and size traits increased, while selection response and heritability gradually decreased. Selection responses were 12.28%-23.35% for harvest weight and 3.58%–13.53% for size traits. Heritability estimates ranged from 0.34±0.09 to 0.48±0.15 for harvest weight and 0.17±0.01–0.38±0.11 for size traits. All phenotypic and genetic parameters differed between various treatments. To conclude, the results demonstrated a potential for mass selection of growth traits in L. vannamei. A breeding scheme could use this information to integrate the effectiveness constituent traits into an index to achieve genetic progress.     

Growth performance of larval and juvenile manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>) from divergently selected individuals of a full-sib family

In this study, the method of divergent selection was employed to test the larval and juvenile growth performance within a full-sib family of Manila clam Ruditapes philippinarum. The 10% largest and 10% smallest clam individuals (on the basis of shell length) of a full-sib family were selected as parents for the fast and slow growing lines, respectively. The difference in shell length was significant among the three lines (fast, control, and slow) tested. The sequence of shell length were fast line > control line > slow line. The responses to selection, realized heritability, and genetic gain were 0.06%–0.81%, 0.04%–0.47% and 0.58%–18.89% in the fast direction, respectively; and were 0.14%–1.27%, 0.08%–0.73%, and 0.31%–49.03% in the slow direction, respectively. The results suggested that there was a large portion of additive genetic variance affecting the growth in the full-sib family. Selection in the fast direction within the full-sib family would greatly improve the growth of R. philippinarum.     

Genetic variation assessed with microsatellites in mass selection lines of the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) in China

Four successive mass selection lines of the Pacific oyster, Crassostrea gigas, selected for faster growth in breeding programs in China were examined at ten polymorphic microsatellite loci to assess the level of allelic diversity and estimate the effective population size. These data were compared with those of their base population. The results showed that the genetic variation of the four generations were maintained at high levels with an average allelic richness of 18.8–20.6, and a mean expected heterozygosity of 0.902–0.921. They were not reduced compared with those of their base population. Estimated effective population sizes based on temporal variances in microsatellite frequencies were smaller to that of sex ratio-corrected broodstock count estimates. Using a relatively large number of broodstock and keeping an equal sex ratio in the broodstock each generation may have contributed to retaining the original genetic diversity and maintaining relatively large effective population size. The results obtained in this study showed that the genetic variation was not affected greatly by mass selection progress and high genetic variation still existed in the mass selection lines, suggesting that there is still potential for increasing the gains in future generations of C. gigas. The present study provided important information for future genetic improvement by selective breeding, and for the design of suitable management guidelines for genetic breeding of C. gigas.     

Mass Selection for Fast Growth in the Third Generation of the Orange Line of the Manila Clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is natively distributed along the Pacific coast of Asia, where it is one of the most important bivalve species for local fisheries. This species has been intentionally introduced due to its high adaptability to various coastal environments and is currently widely cultivated in Europe and North America. We have initiated a selective breeding program to improve the growth rate of the Manila clam since 2007, and a full-sib family with orange shell color was selected. This family features high survival but slow growth. Thereafter, two generations of mass selection were conducted in this family by 10% upward selection for faster growth. In 2011, three types of lines were produced by selecting the breeder from the second-generation of selected lines. These lines are SS(subject to three generations of selection for growth in shell length), SC(selected for the second but not the third generation), and C(randomly sampled individuals from the national population). The genetic parameters of the shell length including hR2(realized heritability), SR(selection response), and GG(genetic gain) were analyzed. Results showed that the shell length of the SS line was significantly larger than those of the SC and C lines at all developmental stages(P 0.05). For the larval stage, the values of hR2, SR, and GG were 0.42, 0.73, and 6.66, respectively. For the juvenile stage, the values of hR2, SR, and GG were 0.40, 0.69, 21.76, respectively. For grow-out stage, the values of hR2, SR, and GG were 0.48, 0.83, and 18.22, respectively. The results of hR2 indicated the good potential of the SS line in selective breeding. The level of GG improvement was encouraging and consistent with the previous expectations. No inbreeding depressions in the shell length were observed in the SS and SC lines at all sampling days. All of the results indicate that maintaining selection pressure in successive generations may be effective in the selective breeding program of this family.     

Linkage Disequilibrium in Wild and Cultured Populations of Pacific Oyster(Crassostrea gigas)

Linkage disequilibrium(LD) can be applied for mapping the actual genes responsible for variation of economically important traits through association mapping.The feasibility and efficacy of association studies are strongly dependent on the extent of LD which determines the number and density of markers in the studied population,as well as the experimental design for an association analysis.In this study,we first characterized the extent of LD in a wild population and a cultured mass-selected line of Pacific oyster(Crassostrea gigas).A total of 88 wild and 96 cultured individuals were selected to assess the level of genome-wide LD with 53 microsatellites,respectively.For syntenic marker pairs,no significant association was observed in the wild population;however,three significant associations occurred in the cultured population,and the significant LD extended up to 12.7 c M,indicating that strong artificial selection is a key force for substantial increase of genome-wide LD in cultured population.The difference of LD between wild and cultured populations showed that association studies in Pacific oyster can be achieved with reasonable marker densities at a relatively low cost by choosing an association mapping population.Furthermore,the frequent occurrence of LD between non-syntenic loci and rare alleles encourages the joint application of linkage analysis and LD mapping when mapping genes in oyster.The information on the linkage disequilibrium in the cultured population is useful for future association mapping in oyster.     

Linkage disequilibrium in wild and cultured populations of Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

Linkage disequilibrium (LD) can be applied for mapping the actual genes responsible for variation of economically important traits through association mapping. The feasibility and efficacy of association studies are strongly dependent on the extent of LD which determines the number and density of markers in the studied population, as well as the experimental design for an association analysis. In this study, we first characterized the extent of LD in a wild population and a cultured mass-selected line of Pacific oyster (Crassostrea gigas). A total of 88 wild and 96 cultured individuals were selected to assess the level of genome-wide LD with 53 microsatellites, respectively. For syntenic marker pairs, no significant association was observed in the wild population; however, three significant associations occurred in the cultured population, and the significant LD extended up to 12.7 cM, indicating that strong artificial selection is a key force for substantial increase of genome-wide LD in cultured population. The difference of LD between wild and cultured populations showed that association studies in Pacific oyster can be achieved with reasonable marker densities at a relatively low cost by choosing an association mapping population. Furthermore, the frequent occurrence of LD between non-syntenic loci and rare alleles encourages the joint application of linkage analysis and LD mapping when mapping genes in oyster. The information on the linkage disequilibrium in the cultured population is useful for future association mapping in oyster.     

马氏珠母贝选系F_2早期选择反应和现实遗传力估计

对马氏珠母贝基础群体进行持续选择建立选系F2,比较了选系F2与对照群体早期生长差别,并估计了选择反应和现实遗传力。结果表明,在第8、14、21和35天,选系F2的平均壳长显著大于对照群体(P<0.05)。在第8、14、21和35天,选择反应和现实遗传力变化范围分别为0.63～0.89和0.36～0.51。本研究表明利用群体选择可以明显改良马氏珠母贝养殖群体的生长性状。     

Optimal content and ratio of lysine to arginine in the diet of Pacific white shrimp, Litopenaeus vannamei

The optimal quantity of dietary lysine (Lys) and arginine (Arg), and the optimal ratio of dietary Lys to Arg for Pacific white shrimp Litopenaeus vannamei were investigated. Coated Lys and Arg were added to a basal diet (37.99% crude protein and 7.28% crude lipid) to provide graded levels of Lys and Arg. The experimental diets contained three Lys levels (2.51%, 2.11%, and 1.70% of total diet), and three Arg levels (1.41%, 1.80%, and 2.21% of total diet) and all combinations of these levels were tested. Pacific white shrimp, with a mean weight of 3.62±0.1 g, were randomly distributed in 36 fiberglass tanks with 30 shrimp per tank and reared on the experimental diets for 50 days. After the feeding trial, the growth performance, survival, feed conversion rate (FCR), body composition and protease and lipase activities in the hepatopancreases of the experimental shrimps were determined. The results show that weight gain (WG), specific growth rate (SGR), FCR, body protein, body Lys and Arg content were significantly affected by dietary Lys and Arg (P <0.05) and improved when dietary Lys and Arg levels were 2.11% ～ 2.51% and 1.80%～2.21%, respectively. Protease and lipase activities in the hepatopancreases of the shrimps appeared higher when dietary Lys and Arg quantities were 2.11% ～2.51% and 1.80%～2.21%, although the difference was not statistically significant (P >0.05). Therefore, according to our results, the optimal Lys and Arg quantities in the diet of Pacific white shrimp, L. vannamei, were considered to be 2.11%–2.51% and 1.80%–2.21%, respectively, and the optimal ratio to be 1:0.88–1:1.05.     

Genome-wide survey and analysis of microsatellites in the Pacific oyster genome: abundance,distribution, and potential for marker development

Microsatellites are a ubiquitous component of the eukaryote genome and constitute one of the most popular sources of molecular markers for genetic studies. However, no data are currently available regarding microsatellites across the entire genome in oysters, despite their importance to the aquaculture industry. We present the fi rst genome-wide investigation of microsatellites in the Pacifi c oyster Crassostrea gigas by analysis of the complete genome, resequencing, and expression data. The Pacifi c oyster genome is rich in microsatellites. A total of 604 653 repeats were identifi ed, in average of one locus per 815 base pairs(bp). A total of 12 836 genes had coding repeats, and 7 332 were expressed normally, including genes with a wide range of molecular functions. Compared with 20 different species of animals, microsatellites in the oyster genome typically exhibited 1) an intermediate overall frequency; 2) relatively uniform contents of(A)n and(C)n repeats and abundant long(C)n repeats(≥24 bp); 3) large average length of(AG)n repeats; and 4) scarcity of trinucleotide repeats. The microsatellite-fl anking regions exhibited a high degree of polymorphism with a heterozygosity rate of around 2.0%, but there was no correlation between heterozygosity and microsatellite abundance. A total of 19 462 polymorphic microsatellites were discovered, and dinucleotide repeats were the most active, with over 26% of loci found to harbor allelic variations. In all, 7 451 loci with high potential for marker development were identifi ed. Better knowledge of the microsatellites in the oyster genome will provide information for the future design of a wide range of molecular markers and contribute to further advancements in the fi eld of oyster genetics, particularly for molecular-based selection and breeding.     

马氏珠母贝黄壳色选育系G_1遗传结构的ISSR分析

挑选11个黄壳色的马氏珠母贝养殖个体为亲本繁殖子代群体,并随机选取50个体为亲本,建立对照群体。采用ISSR分子标记对马氏珠母贝黄壳色群体和对照群体的遗传结构差异进行分析。结果表明,黄壳色和对照群体的遗传多样性分别为0.358 7和0.373 3,多态位点比例分别为86.89%和90.16%;群体间遗传分化系数为3.75%。可见,经过一代选择后,黄壳色群体遗传结构指标略低于对照群体,但两个群体未发生明显分化。     

Pond culture of seaweed Sargassum hemiphyllum in southern China

The seaweed Sargassum hemiphyllum is widely distributed throughout the coastal waters of Asia and has high commercial value.In recent years,its natural biomass has declined due to over-exploitation and environmental pollution.To seek for a feasible way to culture this seaweed efficiently,we designed a simple long-line system in a shrimp pond for the culture during winter,and the growth and nutritional composition of the seaweed were examined.Results show that the culture system was durable and flexible allowing S.hemiphyllum to grow vertically off the muddy bottom of the pond.Although the length of pondcultured S.hemiphyllum was inhibited by water depth,the weight-specific growth rate((1.65±0.17)%/d) was nearly three times higher than that of wild plants((0.62±0.19)%/d).The crude protein(6.92%±0.88%) and ash content(21.52%±0.07%) of the pond-cultured seaweed were significantly lower than those of the wild plants(9.38%±0.43% and 26.93%±0.07%,respectively);however,crude fat(1.01%±0.04%) was significantly higher than that of the wild plants(0.87%±0.02%).In addition,the nutritional composition of both pond-cultured and wild S.hemiphyllum was comparable to or even higher than those of other common seaweeds being used as food and/or aquaculture fodder.Future studies shall be focused on the impact of environmental parameters on its growth and nutritional composition.     

A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.     

Early embryo and larval development of inviable intergeneric hybrids derived from <Emphasis Type="Italic">Crassostrea angulata</Emphasis> and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>

To detect the intergeneric hybridization between the oyster Crassostrea angulata and Saccostrea cucullata coexisting along the southern coast of China, reciprocal crosses were conducted between the two species. Barriers for sperm recognizing, binding, penetrating the egg, and forming the pronucleus were detected by fluorescence staining. From the results, although fertilization success was observed in hybrid crosses, the overall fertilization rate was lower than that of intraspecific crosses. A large number of hybrid larvae died at 6–8 d after hatching, and those survived could not complete metamorphosis. C. angulata ♀× S. cucullata ♂ larvae had a growth rate similar to that of the maternal species, whereas S. cucullata ♀ × C. angulata ♂ larvae grew the slowest among all crosses. Molecular genetics analysis revealed that hybrid progeny were amphimixis hybrids. This study demonstrated that hybrid embryos generated by crossing C. angulata and S. cucullata could develop normally to the larval state, but could not complete metamorphosis and then develop to the spat stage. Thus, there is a post-reproductive isolation between C. angulata and S. cucullata.     

Production of polyhydroxybutyrate by the marine photosynthetic bacterium Rhodovulum sulfidophilum P5

The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of the marine photosynthetic bacterium Rhodovulum sulfidophilum P5 under aerobic-dark conditions. The results show that the accumulation of PHB in strain P5 is a growth-associated process. Strain P5 had maximum biomass and PHB accumulation at 2%-3% NaCl, suggesting that the bacterium can maintain growth and potentially produce PHB at natural seawater salinity. In the nitrogen source test, the maximum biomass accumulation (8.10±0.09 g/L) and PHB production (1.11±0.13 g/L and 14.62%±2.25% of the cell dry weight) were observed when peptone and ammonium chloride were used as the sole nitrogen source. NH 4 + -N was better for PHB production than other nitrogen sources. In the carbon source test, the maximum biomass concentration (7.65±0.05 g/L) was obtained with malic acid as the sole carbon source, whereas the maximum yield of PHB (5.03±0.18 g/L and 66.93%±1.69% of the cell dry weight) was obtained with sodium pyruvate as the sole carbon source. In the carbon to nitrogen ratios test, sodium pyruvate and ammonium chloride were selected as the carbon and nitrogen sources, respectively. The best carbon to nitrogen molar ratio for biomass accumulation (8.77±0.58 g/L) and PHB production (6.07±0.25 g/L and 69.25%±2.05% of the cell dry weight) was 25. The results provide valuable data on the production of PHB by R. sulfidophilum P5 and further studies are on-going for best cell growth and PHB yield.     

Relationship Between Shell Color and Growth and Survival Traits in the Pacific Oyster Crassostrea gigas

Shell color is regarded as an economic trait in many breeding programs of bivalve mollusks, but the relationship between shell color and production traits remains controversial. In our breeding program of Crassostrea gigas, solid white, black, and orange shell lines were obtained, and second-generation(F2) and testcross families with segregating shell colors were constructed by crossing these three lines. These segregating families provided ideal samples for investigating the relationship between shell color and production traits in C. gigas. The growth and survival of 7-month-old oysters with different shell colors sampled within the same families were compared in seven F2 families and 13 testcrosses. In addition, the growth and survival of oysters from the three shell color lines were compared at 4 and 16 months of age. The growth and survival rates of the orange shell line were significantly lower than those of the white and black shell lines. However, no significant difference in growth between oysters with different shell colors was observed within segregating families, except the testcrosses produced by crossing orange-and white-shelled grandparents, and no significant difference in survival was observed in any family. Overall, no significant correlation was observed between shell color and production traits in C. gigas. These results suggest that shell color cannot be used as a marker to guide the selection of growth and survival traits. Thus shell color and production traits should be selected independently in oyster breeding programs.     

ZResponse to selection, heritability and genetic correlations between body weight and body size in Pacific white shrimp, Litopenaeus vannamei

To quantify the response to selection,heritability and genetic correlations between weight and size of Litopenaeus vannamei,the body weight (BW),total length (TL),body length (BL),first abdominal segment depth (FASD),third abdominal segment depth (TASD),first abdominal segment width (FASW),and partial carapace length (PCL) of 5-month-old parents and of offspring were measured by calculating seven body measurings of offspring produced by a nested mating design.Seventeen half-sib families and 42 full-sib families ofL.vannamei were produced using artificial fertilization from 2-4 dams by each sire,and measured at around five months post-metamorphosis.The results show that heritabilities among various traits were high:0.515士0.030 for body weight and 0.394士0.030 for total length.After one generation of selection,the selection response was 10.70% for offspring growth.In the 5"’ month,the realized heritability for weight was 0.296 for the offspring generation.Genetic correlations between body weight and body size were highly variable.The results indicate that external morphological parameters can be applied during breeder selection for enhancing the growth without sacrificing animals for determining the body size and breed ability;and selective breeding can be improved significantly,simultaneously with increased production.     

Cytogenetic mechanism for the aneuploidy and mosaicism found in tetraploid Pacific oyster Crassostrea gigas (Thunberg)

Chromosome constitution was investigated in adult tetraploid Pacific oyster produced by blocking the first polar body of triploid eggs which were fertilized with haploid sperms. A high incidence of aneuploid and heteroploid mosaics were found among the offspring. Of 20 individuals identified, only 9 (45%) were eutetraploid which contained 40 chromosomes; 2 (10%) were aneuploid (hypotetraploid), which contained 39 and 38 chromosomes, respectively; and 9 (45%) were heteroploid mosaics. One mosaic was consisted of cells containing 40 and 39 chromosomes, respectiovely (1:1 in cell number), while the other 8 were consisted of cells containing chromosomes varying between tetraploid and triploid. It was also interesting to note that 3 mosaics even contained some diploid cells with 20 chromosomes. A certain number of cells of 2 tetraploids and 8 mosaics spread with 32–37 well-scattered and some clumped chromosomes at metaphase. The percentage of aneuploid cells with chromosomes varying between triploid and tetraploid correlated significantly with that of heteroploid mosaics cells with clumping chromosomes (P<0.05). Our findings suggested that reversion existed in both tetraploid and triploid oyster and chromosome clumping may underline the chromosome elimination in tetraploid oyster. It seems that the reversing cells, at least some of them, continuously eliminate their chromosomes until the most stable diploid state is established.     

Inheritance of 15 microsatellites in the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping. Supported by the National Natural Science Foundation of China (NO.40730845, 39825121)     

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas： segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.     

Effect of substrate component on the growth and survival of juvenile sunray surf clam (<Emphasis Type="Italic">Mactra chinensis</Emphasis> Philippi)

Substrate is a critical environmental factor affecting the activity of bivalves. To examine the effect of the substrate component on the growth and survival of juvenile sunray surf clam(Mactra chinensis Philippi), a series of short-term experiments were conducted using a variety of substrates with different ratios of sand to mud. The experimental group cultured without substrate showed poor survival, with all juveniles died after day 20. The juveniles cultured in mud without sand showed a lower survival rate(25.54% ± 0.40% on day 45) than those in other groups. The juveniles cultured in sand without mud, or the mixtures of sand and mud with a ratio of 1:1 and 2:1, respectively, exhibited modest survival at day 45. Maximal weight gain rate(WGR), shell length growth rate(LGR), and specific growth rate(SGR) were observed when the sand concentration was 61.97%, 77.69%, and 64.64%, respectively. As the fast growth and high survival were observed when the sand to mud ratio was 1:1(50% sand) and 2:1(67% sand), a sand concentration of more than 50% is optimal. The optimal concentration of sand in the substrate for rearing juvenile sunray surf clams was 67% which resulted in the fastest growth and highest survival. These results can be used to developing a nursery/farming technique of improving the yield of sunray surf clams.