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Simple Summary: In this study, we performed a simplified genome sequencing of 301 individuals from 10 populations of Ruditapes philippinarum in coastal China using restriction site-associated DNA sequencing (RAD-seq) technology, and obtained a large number of single-nucleotide polymorphism (SNP) markers of Manila clam. Eighty-eight SNP markers were successfully developed using high-resolution melting (HRM) analysis, and the genetic structure and genetic diversity of two populations were analyzed. SNP markers provide a valuable resource for population and conservation genetics studies of this commercially important species. Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam Ruditapes philippinarum using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of FIS was estimated to be from −0.9643 to 1.0000. The global Fst value was 0.1248 (p < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy–Weinberg equilibrium (p < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species. [ABSTRACT FROM AUTHOR]

Simple Summary: China has the largest production yield of Manila clam Ruditapes philippinarum in the world. Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation and inbreeding depression. In this paper, we investigate the genetic diversity and differentiation of R. philippinarum populations. The present results not only provide the scientific basis for the phylogenetic relationship of clam resources, but also lay the foundation for natural population conservation and genetic improvement of R. philippinarum. China has the largest production yield of Manila clam Ruditapes philippinarum in the world. Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation and inbreeding depression. To understand the genetic and haplotype diversity of R. philippinarum, 14 clam populations sampled from different regions of China were analyzed by three molecular markers, including COI, 16SrRNA and ITS. Based on the results of the COI and ITS genes, the 14 populations showed a moderate to high level of genetic diversity, with an average haplotype diversity of 0.9242 and nucleotide diversity of 0.05248. AMOVA showed that there was significant genetic differentiation among all populations (mean FST of the total population was 0.4534). Pairwise FST analysis showed that genetic differentiation reached significant levels between Laizhou and other populations. Two Laizhou populations showed great divergence from other populations, forming an independent branch in the phylogenetic tree. The shared haplotypes Hap_2 and Hap_4 of COI appeared most frequently in most clam populations. In contrast, 16SrRNA analysis of the clam populations revealed the dominated haplotype Hap_2, accounting for 70% of the total number of individuals. The haplotype diversity of the Laizhou population (Laizhou shell-wide (KK) and Laizhou dock (LZMT)) was relatively higher than other populations, showing multiple unique haplotypes (e.g., Hap_40, Hap_41 and Hap_42). These findings of genetic and haplotype diversity of clam populations provide guiding information for genetic resource conservation and genetic improvement of the commercially important R. philippinarum. [ABSTRACT FROM AUTHOR]

Simple Summary: The Manila clam (Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. The increasing expanding of clam culture may result in some serious problems. In this paper, we investigated the genetic diversity and differentiation of R. philippinarum populations and tested the hypothesis that clam population differentiation is influenced by the southern breeding and northern culture. The present findings will provide useful information for natural resource conservation and genetic breeding of the Manila clam in China. The Manila clam (Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. With the continuous expansion of clam farming scale, it may lead to some serious problems, including loss of genetic variation, inbreeding depression, and reduced effective population size (Ne). In the present study, eleven microsatellite markers were used to investigate the genetic diversity and differentiation among 13 clam populations along the coast of China. As a result, 150 alleles were detected according to the genotyping results of eleven microsatellite loci. The observed heterozygosity (Ho) was estimated to be ranging from 0.437 to 0.678, while the expected heterozygosity (He) was calculated to be varying from 0.587 to 0.700. Fst values between populations ranged from 0.0046-0.1983. In particular, the Laizhou population had the highest genetic variability, which was significantly different from the others (all Fst values > 0.1). For all the clam populations, there was no significant linear regression between genetic and geographic distance, indicating that these populations do not follow a pattern of isolation by distance (IBD). Genetic structure was estimated according to NJ, principal coordinates (PCoA), and structure-based clustering. Estimates of effective population size range from dozens to thousands among different populations, based on linkage-disequilibrium and molecular coancestry methods. The results reveal the genetic diversity of clams and verify the hypothesis that clam population differentiation may be influenced by the mode of southern breeding and northern culture, providing guiding information for natural resource conservation and genetic breeding of clams. [ABSTRACT FROM AUTHOR]

Knowledge of early development in bivalves is of great importance to understand the function of animal organ systems and the evolution of phenotypic diversity. Manila clam (Ruditapes philippinarum) is an economically important bivalve living in marine intertidal zones. To determine the pattern of muscle development in the clams, we investigate the characteristics of musculature development utilizing phalloidin staining and confocal microscopy. Myofilaments first appear at the early trochophore stage, and gradually become orderly arranged during the transition from trochophore to veliger. For veliger, larval muscle system is mainly composed of dorsal velum retractors, medio-dorsal velum retractors, ventral velum retractors, ventral larval retractors and anterior and posterior adductor muscles. After metamorphosis, the muscle system of late veliger has been reconstructed, showing the irreversible shrink of velum retractor muscles, the rapid growth of wedge-shaped foot and mantle margins. One of the most striking changes in settled spats is the development of sophisticated architecture of foot musculature, which consists of transverse pedal muscles, anterior foot retractor and posterior foot retractor. These findings will not only provide the basis to understand the dynamic pattern of myogenesis in the burrowing bivalves, but also provide valuable information for comparative analysis of muscle development among bivalves. [Display omitted] • Myogenesis in clams represents the similar pattern of muscle remodeling in bivalves. • There is a partial degeneration of the anterior adductor in clams, in contrast to oysters and scallops. • The sequential development of sophisticated foot musculature has been observed in clams. • The present findings on myogenesis in clams may strengthen our understanding of muscle evolution in bivalves. [ABSTRACT FROM AUTHOR]

Hybridization is an important breeding method to improve productive traits in aquaculture. The Manila clam Ruditapes philippenarum , a worldwide cultured bivalve, has achieved the great commercial success owing to a global appreciation of its delicious taste and affordable price. In order to evaluate the aquaculture potential of clam hybridization, a complete diallel cross between two geographical populations (Shandong and Guangdong) was performed in the present study. Two intra-population mating groups LL (L♀ × L♂) and ZZ (Z♀ × Z♂) and two crossbreeding groups ZL (Z♀ × L♂) and LZ (L♀ × Z♂) were established to investigate the potential heterosis of growth and survival in the clams. Three full-sib families were constructed in each mating group to calculate the growth and survival heterosis. During the larval stage (2 ∼ 10d), no significant difference was detected in averaged shell length between reciprocal hybrids and inbred groups. In contrast, hybrid spats after 15d cultivation showed a significantly larger size (shell length and shell height) than that of inbred spats. For the reciprocal hybrids, the greatest growth heterosis of shell length and height (33.42% and 33.13%) was detected from 20 days after fertilization in ZL. After 40d cultivation, ZL displayed the most growth potential in shell length and height, reaching maximum values of 0.900 and 0.858 mm, respectively. The hybrids had 25% ∼ 40% higher survival rates than those of the inbred groups, with the highest survival heterosis in ZL. The positive values for the general combining ability (GCA) were consistently detected in the Zhanjiang population. At day 40, the special combining ability (SCA) in ZL was approximately two-fold higher than that in LZ. These findings indicate that the hybrids produced by Laizhou males and Zhanjiang females have the greater aquaculture potential, providing a useful crossbreeding strategy for clam genetic improvement in aquaculture. • Intraspecific crossbreeding of clams produced hybrids with great growth potential. • The growth and survival heterosis were found in larvae and spats. • The greatest growth heterosis was detected at 20d after fertilization. • Crossbreeding of Laizhou males and Zhanjiang females has the great aquaculture potential. [ABSTRACT FROM AUTHOR]

• The effect of hypoxia on clam ruditapes philippinarum was evaluated in many aspects. • The prolongation of hypoxia stress at DO 2.0 mg/L can cause the irreversible damages of cellular structures in clams. • The expression levels of genes related to energy metabolism was significantly affected by the hypoxia stress. • The extent of hypoxia impacts on marine bivalves may be underestimated in the coastal areas. Hypoxia has become one of the major environmental problems in the aquaculture industry. As one of the most commercially important bivalves, Manila clam Ruditapes philippinarum may be suffering substantial mortality attributable to hypoxia. The physiological and molecular responses to hypoxia stress in Manila clam were evaluated at two levels of low dissolved oxygen: 0.5 mg/L (DO 0.5 mg/L) and 2.0 mg/L (DO 2.0 mg/L). With the prolongation of hypoxia stress, the mortality rate was 100% at 156 h under DO 0.5 mg/L. In contrast, 50% of clams survived after 240 h of stress at DO 2.0 mg/L. After the hypoxia stress, some severe structural damages were observed in gill, axe foot, hepatopancreas tissues, such as cell rupture and mitochondrial vacuolization. For the hypoxia-stressed clams, the significant rise and decline of enzyme activity (LDH and T-AOC) was observed in gills, in contrast to the reduction of glycogen content. Furthermore, the expression levels of genes related to energy metabolism (SDH, PK, Na+/ K +-ATPase, NF-κB and HIF-1α) was significantly affected by the hypoxia stress. It is therefore suggested that the short-term survival of clams under hypoxia may be dependent on stress protection by antioxidants, energy allocation, and tissue energy reserves (such as glycogen stores). Despite this, the prolongation of hypoxia stress at DO 2.0 mg/L may cause the irreversible damages of cellular structures in clam tissues, eventually leading to the death of clams. We therefore support the hypothesis that the extent of hypoxia impacts on marine bivalves may be underestimated in the coastal areas. [ABSTRACT FROM AUTHOR]

Mudflat shellfish have evolved well-adapted strategies for coping with dynamic environmental fluxes and stressful conditions, including oxygen availability. The Manila clams Ruditapes philippinarum are worldwide cultured shellfish in marine intertidal zone, which usually encounter great risk of acute hypoxia exposure in coastal habitats. To reveal the effects of acute hypoxia on metabolic changes of the clams, we performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites during acute hypoxia stress at the whole-organism level. The comparative transcriptome analysis reveals that the clams show the remarkable depression in a variety of biological performance, such as metabolic rates, neuronal activity, biomineralization activity, and cell proliferation and differentiation at the hypoxic condition. The metabolomic analysis reveals that amino acid metabolism plays a critical role in the metabolic changes of the clams in response to acute hypoxia. A variety of free amino acids may not only be served as the potential osmolytes for osmotic regulation, but also may contribute to energy production during the acute hypoxia exposure. The metabolite analysis also reveals several important biomarkers for metabolic changes, and provides new insights into how clams deal with acute hypoxia. These findings suggest that clams may get through acute hypoxia stress by the adaptive metabolic strategy to survive short-period of acute hypoxia which is likely to occur in their typical habitat. The present findings will not only shed lights on the molecular and metabolic mechanisms of adaptive strategies under stressful conditions, but also provide the signaling metabolites to assess the physiological states of clams in aquaculture. [Display omitted] • Metabolomics and transcriptomics were applied to reveal molecular responses of the clams to acute hypoxia. • The clams survive short-period of acute hypoxic stress by the remarkable depression in biological process. • Amino acids may not only be served as potential osmolytes, but also contribute to energy production in response to hypoxia. • The metabolic adaptation to hypoxia is regulated by starch and sucrose metabolism, carnitine metabolism, and sphingolipid. [ABSTRACT FROM AUTHOR]