Identification of Shell Color-Associated Genes Using Mantle Branch-Specific RNA Sequencing of Yellow-Colored Line of Pearl Oyster Pinctada fucata martensii

The yellow-colored line of pearl oyster Pinctada fucata martensii displays a yellow prismatic layer and a white nacreous layer that can be used as an ideal model for research on shell color formation.Micro-Raman spectroscopy and transcriptome analyses were performed to explore the potential molecular mechanism underlying the phenotype differentiation.The micro-Raman spectroscopy results indicate that the prismatic layer exhibits distinct characteristic peaks of carotenoids,while these peaks are not prominent in the nacreous layer.In the transcriptome comparison of the central zone of mantle and mantle edge tissue,which function in nacreous and prismatic layer formation,respectively,935 significantly differentially expressed genes(DEGs)were identified,with 385 genes upregulated and 227 genes downregulated(|log_(2)(Fold change)|>1 and false discovery rate<0.05)in the mantle edge tissue.Among these genes,some were associated with melanoma/melanogenesis,such as tyrosinase,zinc metalloprotease,glutathione S-transferase,and ATP-binding cassette sub-family;some were associated with the carotenoid-related pathway,including scavenger receptors,cytochrome P450 and lipoprotein receptor.Genes associated with porphyrin metabolism,including porphobilinogen deaminase,and copper/zinc superoxide dismutase,and genes associated with shell matrix protein,including amorphous calcium carbonate binding protein,shematrin,PIF,and collagen,also exhibited significantly different expressions.It is speculated that the different colours between prismatic layer and nacreous layer in the yellow-colored line of P.f.martensii might be resulted from melanin,carotenoids and porphyrin metabolism,while genes related to shell structure and biomineralization might also affect coloration.Our results provide new insights to understand the mechanism of shell color formation in mollusca.

Comparison of growth and survival of larvae among different shell color stocks of bay scallop Argopecten irradians irradians(Lamarck 1819)

In order to improve production and breed new broods of bay scallop Argopecten irradians irradians, different-colored orange, purple and white lines were established by two mating methods of self-fertilization and mass spawning at a commercial scallop hatchery in spring, 2002. And then larval growth and survival of different lines was compared to test whether there is a relationship between shell color and growth and survival at early developmental stage. Both growth and survival have no significant differences among different experimental larvae of self-fertilization or mass spawning. Results are as following in the order of orange, purple and white shell stock. For the self-fertilization, growth rates of larvae were 6.174, 6.412, and 6.599 μm/d, respectively. Survival rates of larvae at Day 3 were 74.41%, 76.86%, and 82.05%; Day 6 were 49.14%, 65.63%, and 52.79%; and Day 9 were 25.06%, 20.80%, and 26.47%, respectively. For the mass spawning, the growth rates were 7.836, 7.941, and 7.878 μm/d, respectively. Survival rates at Day 3 were 93.05%, 91.95%, and 92.50%; Day 6 were 79.17%, 78.05%, and 82.50%; and Day 9 were 34.72%, 36.67%, and 38.33%, respectively. The absence of any relationship between shell color and growth and survival at the larval stage may be resulted from their common genetic basis.

UPLC-MS metabolomics provides insights into the diff erences between black- and white-shelled Pacifi c oysters Crassostrea gigas

A variety of shell colors are one of the most fundamental characteristics of molluscs,which have importantly ecological and economic signifi cance.The Pacifi c oyster Crassostrea gigas is distributed in many sea areas around the world and also an aquacultured mollusc with high nutritional value.In this study,the whole soft body and the mantle tissue of black-shelled Pacifi c oyster(BSO)and white-shelled Pacifi c oyster(WSO)with starkly diff erent melanin contents were compared,and the diff erences in physiology and metabolism between BSO and WSO were analyzed.The results of physiological indicators suggested BSO show more melanin,more dry matter,more crude lipid content,and stronger ability to scavenge free radicals than WSO.The altered metabolites of glycerophospholipids,fatty acyls,and steroids revealed diff erent regulatory mechanisms of lipids.The correlation analysis of metabolomics and previously published RNAseq data suggested that BSO and WSO mainly diff ered in the basal metabolic processes,such as lipid,amino acid and purine metabolisms.This study provides insights into the changes in the physiological indictors and the metabolites of oysters with varying melanin content.

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.

De novo transcriptome sequencing reveals candidate genes involved in orange shell coloration of bay scallop Argopecten irradians

Molluscan shell color has received persistent attention for its distinctive diversity and complexity. In the present study, six transcriptome libraries obtained from two developmental stages, pre-pigmentation and post-pigmentation, were used for paired-end sequencing in the bay scallop Argopecten irradians. In total, 289 839 646 paired-end reads were assembled into 70 929 transcripts. Using BLASTX and BLASTN, 30 896 unigenes were successfully annotated against the SWISS-PROT, NR, and KOG database. Gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes classification identified numbers of unigenes involved in biomineralization and pigmentation. Digital gene expression analysis revealed that melanin, trace metal elements and porphyrins are potentially involved in shell coloration of A. irradians.

Characteristics of a Novel Tyrosinase Gene Involved in the Formation of Shell Color in Hard Clam Meretrix meretrix

Hard clam(Meretrix meretrix)has a rich shell color variation among individuals from light yellow to bluish gray,brown red,and black,which may associating with melanin.Tyrosinase(Tyr)is a key rate-limiting enzyme for the biosynthesis of melanin,which affects the dark color of animal skin,hair,fur,scales and feathers.Here,we isolated and characterized the full-length cDNA sequence of M.meretrix tyrosinase gene(MmTyr)which encodes a protein with 689 amino acids.Sequence characteristics and phylogenetic analysis showed that there are a variety of Tyr genes in mollusks,which can be divided into different categories.The abundance of MmTyr transcript was detected in six tissues including mantle,adductor muscle,digestive gland,foot,gill and siphon of adult M.meretrix with qRT-PCR.The results showed a higher expression specifically in the mantle,digestive gland and siphon,which was consistent with the phenotypic color difference.In the embryos and larvae at different developmental stages,high expression was found in the trochophore larvae and juvenile clams.Among three M.meretrix strains with different shell colors,the expression level of MmTyr and total melanin content in the mantle edge both showed significant differences,suggesting that MmTyr is involved in the formation of shell color.The results obtained in this study will improve our understanding of molluscan tyrosinase gene function and molecular mechanism of shell color pattern determination.

Growth performance of the clam, Meretrix meretrix, breeding-selection populations cultured in different conditions

A breeding-selection program for the clam, Meretrix meretrix, was conducted since 2004. Two of the selec- tion populations were generated with the shell color pattern as an additional selection criterion and named as SP （purple stripes） population and SB （black dots） population. The third-generation SP and SB popula- tions （08G3SP and 08G3SB, respectively） were cultured at two commercial clam farms and a nursery pond and their shell lengths were compared. 08G3SB clams had significantly larger sizes than 08G3SP clams at commercial clam farms （p 〈 0.05）, yet 08G3SB individuals were significantly smaller than 08G3SP individ- uals at the nursery pond （p 〈 0.05）. Then, we examined the growth of the fourth-generation SP and SB populations （10G4SP and 10G4SB, respectively） at a commercial farm, and found that the shell lengths of the 10G4SB clams increased at a significantly higher growth rate than the 10G4SP clams （p 〈 0.05） from May to September, when the water temperature was between 24.2-27.5℃, while 10G4SB lost the growth advantage in the other months. These results indicated that SP and SB populations responded differently to environmental factors, so it is beneficial for the clam farmers to select a suitable population according to the culture environment. Furthermore, a diallel mating of the SB and SP populations was designed to investigate whether their hybrid population would show heterosis. However, the heterosis was not shown in this study, which might result from the slight genetic divergence between SB and SP populations.