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The mammalian target of rapamycin (mTOR) has been shown to play a central role in regulating cell growth and metabolism. However, little is known about the function of mTOR in nutrient metabolism in bivalve mollusks. In this study, the role of mTOR in the regulation of nutrient metabolism was investigated in Sinonovacula constricta. First, the activation of mTOR was assayed after starvation and refeeding. Afterwards, the role of mTOR in the regulation of nutrient metabolism was investigated using an activator (MHY1485) or inhibitor (rapamycin) of mTOR. The open reading frame of the S. constricta mTOR is 7416 bp in length and encodes a polypeptide consisting of 2471 amino acids. The mTOR amino acid sequence of S. constricta was highly conserved when compared with other species and had a close evolutionary relationship with the TOR proteins of Crassostrea gigas and Lingula anatine. mTOR was expressed in the intestine, exhalent siphon, labial palppus, muscle, inhalent siphon, gill, mantle, digestive land, and gonad tissue of S. constricta, with the highest expression in muscle. During starvation, the level of phosphorylated mTOR protein was relatively low, and the ratio of LC3II/LC3I protein and the AMPKα mRNA level significantly increased with the increase in starvation time. After feeding, the level of phosphorylated mTOR protein increased from 0.13 to 0.56, and the ratio of LC3II/I protein and AMPKα mRNA level decreased from 1.17 to 0.38. MHY1485 significantly increased the level of phosphorylated 4E-BP1 and significantly decreased the ratio of LC3II/I proteins. Furthermore, MHY1485 significantly increased the mRNA level of the glucose metabolism-related gene glucokinase (GK), significantly decreased the mRNA expression of the G6P gene, and significantly increased the mRNA expression of the lipid synthesis-related genes sterol-regulatory element-binding protein (SREBP) and stearoyl-CoA desaturase (SCD). Rapamycin significantly reduced the level of phosphorylated 4E-BP1 and the mRNA expression of mTOR, and the expression level of phosphorylated 4EBP1 decreased from 0.97 to 0.28. Meanwhile, it also significantly reduced the mRNA expression of glucose metabolism-related genes GK, pyruvate kinase (PK), glucose transporter 1 (GLUT1), and G6P, as well as lipid synthesis-related genes SCD and acetyl-CoA carboxylase (ACC). These results indicate a conserved role of mTOR in regulating nutritional metabolism, including glucose metabolism, lipid synthesis, and autophagy in S. constricta. [ABSTRACT FROM AUTHOR]

The razor clam, Sinonovacula constricta, contains high levels of long-chain PUFA (LC-PUFA), which are critical for human health. In addition, S. constricta is the first marine mollusc demonstrated to possess Δ6 fatty acyl desaturase (Fad) and complete LC-PUFA biosynthetic ability, providing a good representative to investigate the molecular mechanism of sterol regulatory element binding proteins (SREBP) in regulating Δ6 Fad for LC-PUFA biosynthesis in marine molluscs. Herein, S. constricta SREBP and Δ6 Fad promoter were cloned and characterised. Subsequently, dual luciferase and electrophoretic mobility shift assays were conducted to explore the SREBP binding elements in the core regulatory region of S. constricta Δ6 Fad promoter. Results showed that S. constricta SREBP had a very conservative basic helix-loop-helix-leucine zipper motif, while S. constricta Δ6 Fad promoter exhibited very poor identity with teleost Fads2 promoters, indicating their differentiation during evolution. A 454 bp region harbouring a core sequence in S. constricta Δ6 Fad promoter was predicted to be essential for the transcriptional activation by SREBP. This was the first report on the regulatory mechanism of LC-PUFA biosynthesis in marine molluscs, which would facilitate optimising the LC-PUFA biosynthetic pathway of bivalves in further studies. [ABSTRACT FROM AUTHOR]

Bivalve lectins perform a crucial function in recognition of foreign particles, such as microalgae and pathogenic bacteria. In this study, a novel C-type lectin form Sinonovacula constricta (ScCL) was characterized. The full-length cDNA of ScCL was 1645 bp, encoding a predicted polypeptide of 273 amino acids with one typical carbohydrate-recognition domain. ScCL has the highest similarity and closest phylogenetic relationship with the C-type lectin from Solen grandis. Real-time PCR analysis showed that ScCL was expressed in all tested tissues, with the highest expression in the foot and the lowest expression in hemocytes. Agglutination activity of ScCL was Ca2+-independent. ScCL showed the strongest agglutination on Chlorella vulgaris , the modest agglutination on Platymonas subcordiformis , Nannochloropsis sp., and Thalassiosira pseudonana , the weakest agglutination on Chaetoceros sp., and no agglutination on Isochrysis zhanjiangensis. Meanwhile, agglutination tests and western blot analysis revealed that the recombinant ScCL protein could agglutinate Staphylococcus aureus and Vibrio harveyi , but could not agglutinate Vibrio anguillarum , Bacillus cereus , or Vibrio parahaemolyticus. Furthermore, ScCL had a high binding activity with LPS and mannose, a low binding activity with LTA, and no binding activity with PGN. The expression of ScCL in the gill of S. constricta fed with C. vulgaris and T. pseudonana was significantly increased at 1 and/or 3 h. After injection with S. aureus , the expression of ScCL in the gill was significantly increased at 3, 6, and 24 h. These results indicated that ScCL was involved in food particle recognition and immunity of S. constricta. • A novel C-type lectin from Sinonovacula constricta (ScCL) was characterized. • ScCL protein could bind to several PAMPs and bacteria. • ScCL protein could agglutinate several microalgae and bacteria in Ca2+-independent. • After feeding Chlorella vulgaris and Thalassiosira pseudonana , or after challenged with staphylococcus aureus , the expression of ScCL was up-regulated. • ScCL may be involved in food recognition and innate immunity. [ABSTRACT FROM AUTHOR]

Bivalves, a highly diverse and the most evolutionarily successful class of invertebrates native to aquatic habitats, provide valuable molecular resources for understanding the evolutionary adaptation and aquatic ecology. Here, we reported a high‐quality chromosome‐level genome assembly of the razor clam Sinonovacula constricta using Pacific Bioscience single‐molecule real‐time sequencing, Illumina paired‐end sequencing, 10X Genomics linked‐reads and Hi‐C reads. The genome size was 1,220.85 Mb, containing scaffold N50 of 65.93 Mb and contig N50 of 976.94 Kb. A total of 899 complete (91.92%) and seven partial (0.72%) matches of the 978 metazoa Benchmarking Universal Single‐Copy Orthologs were determined in this genome assembly. And Hi‐C scaffolding of the genome resulted in 19 pseudochromosomes. A total of 28,594 protein‐coding genes were predicted in the S. constricta genome, of which 25,413 genes (88.88%) were functionally annotated. In addition, 39.79% of the assembled genome was composed of repetitive sequences, and 4,372 noncoding RNAs were identified. The enrichment analyses of the significantly expanded and contracted genes suggested an evolutionary adaptation of S. constricta to highly stressful living environments. In summary, the genomic resources generated in this work not only provide a valuable reference genome for investigating the molecular mechanisms of S. constricta biological functions and evolutionary adaptation, but also facilitate its genetic improvement and disease treatment. Meanwhile, the obtained genome greatly improves our understanding of the genetics of molluscs and their comparative evolution. [ABSTRACT FROM AUTHOR]

BACKGROUND Sinonovacula constricta is an economically and nutritionally important bivalve native to the estuaries and mudflats of China, Japan and Korea. In the present study, S. constricta, cultured either under experimental conditions or collected directly from natural coastal areas with different seawater salinities, was investigated for changes in proximates, amino acids and lipids. RESULTS When culture salinity was increased, levels of moisture, carbohydrate, crude protein and crude lipid were significantly decreased, whereas the level of ash was significantly increased. The level of Ala was increased by 1.5- to 2-fold, whereas the contents of most lipids were significantly decreased, and the proportion of phosphatidylethanolamine was significantly increased. Notably, a high proportion of ceramide aminoethylphosphonates was detected in S. constricta reared at all salinities. The energy content appears to be higher in S. constricta reared at higher salinity. In experimental S. constricta, when the salinity was enhanced, the changes of compositions were very close to those reared at constant high salinity. CONCLUSION Sinonovacula constricta reared at higher salinities possesses a superior quality. A short period of exposure to a higher salinity for farmed S. constricta reared at a lower salinity before harvest would be useful with respect to improving its nutritive value. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta , which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the 'Sprecher pathway'. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs. • The S. constricta Elovl involved in LC-PUFA synthesis were investigated. • The desaturation ability of S. constricta Δ6 Fad toward 24:5n-3 was determined. • S. constricta is the first mollusc with capacity to operate the Sprecher pathway. • DHA biosynthetic ability was limited to certain marine molluscs. [ABSTRACT FROM AUTHOR]

Melatonin affects physiological processes that have circadian and seasonal cycles and causes behavioural changes in nearly all organisms, ranging from bacteria to mammals. However, no studies have focused on the presence, biosynthetic pathway, and rhythms of melatonin in bivalves, which are considered as the class of molluscs with the highest economic value. Here, the metabolic pathway and tissue distribution of melatonin, tissue transcription profiles of melatonin-related genes [key synthases arylalkylamine N-acetyltransferase type 2 and 5 (aanat2 , aanat5), N-acetylserotonin methyltransferase (asmt), and melatonin receptor type 1 A1 and C3 (MTNR1A1 , MTNR1C3)], daily changes in melatonin, transcripts of genes, and effects of four lights (blue, cyan, orange, and red) during the night on melatonin secretion were studied in a bivalve species, razor clam Sinonovacula constricta. Our results showed the presence of complete metabolic and circadian entrainment pathways of melatonin. The melatonin content in the lymphocytes and labial palp was higher than that in the other studied tissues, and aanat2 , aanat5 , asmt , MTNR1A1 , and MTNR1C3 mRNA levels were the highest in the labial palp. Melatonin fluctuated rhythmically in lymphocytes and labial palps and increased at night. Similarly, aanat2 , aanat5 , asmt , MTNR1A1 , and MTNR1C3 mRNA levels were rhythmic in the labial palp, peaking at night. In addition, blue light effectively inhibited the nocturnal secretion of melatonin in the clam. In summary, our results suggest that melatonin plays a role in light signal transduction and the circadian system in S. constricta. • Melatonin could be biosynthesized by the clam Sinonovacula constricta. • Labial palp probably is an important tissue where melatonin exerted its function. • The biosynthesis and secretion of melatonin in the clam are enhanced under the dark phase of the photoperiod. • Blue light inhibits melatonin production in the clam. [ABSTRACT FROM AUTHOR]

In the present study, we explored the effects of seven light treatments (white, violet, blue, cyan, green, yellow, and red) on juvenile Sinonovacula constricta (length, 693.79 ± 82.91 μm), employing dark treatment as a control. A rearing experiment was conducted for one week in which the feeding rate was examined daily, e.g., one, two and three h after feeding. At the conclusion of the experiment, overall growth was calculated, and enzymatic activity related to digestion, superoxide dismutase, catalase, and glutathione peroxidase pertaining to antioxidant biomarkers, as well as expression levels of genes related to antioxidation and stress response, were examined. The results showed that the highest growth rates were observed in the yellow light group, while the lowest growth rates were found in the violet and blue light groups; on the other hand, no significant differences were found among the other groups. Meanwhile, when exposed to white, yellow, or green light, juveniles exhibited high feeding rates, while those in the violet and blue light groups showed low feeding rates. Regarding enzymatic activity related to digestion, those of α -amylase, lipase, pepsin, and trypsin were relatively higher in the yellow light group than in the other groups. Regarding oxidative stress, the yellow light group showed higher superoxide dismutase, catalase, and glutathione peroxidase activities than the other groups. In contrast, the lowest malonaldehyde level was found in the white light group, followed by yellow light, while there were no significant differences among the other groups. In addition, the expression levels of genes related to antioxidation and stress response (heat shock protein 70 and glucose-regulated protein 78) in juveniles generally exhibited higher levels in the red light group, whereas they showed relatively lower levels in the yellow light group. Collectively, these results suggest that the application of yellow light may facilitate the growth of juvenile S. constricta , while red and violet light have opposite effects. • Light spectrum differently influenced the growth of juvenile S. constricta. • Yellow light facilitated the growth of juvenile S. constricta. • Yellow light induced less stress to S. constricta. • Red and violet lights impaired the growth of juvenile S. constricta. [ABSTRACT FROM AUTHOR]

Sinonovacula constricta is an important bivalve with strong photosensitivity and a robust circadian rhythm. To elucidate the light entrainment of the molecular circadian clock in this species, this study identified the clock genes, characterized their tissue distribution, and investigated their transcriptional variations under different light regimes: LD (12 h light/12 h dark cycle), LL (continuous light), and DD (continuous dark). The results showed that the S. constricta clock system consists of seven genes [ Circadian locomotor output cycles kaput (ScClock), Brain and muscle ARNT-like protein (ScBmal), Cryptochrome (ScCry1 and 2), Period (ScPer), and Timeless (ScTimeless1 and 2)] that are closely related to their orthologs in marine mollusks and contained corresponding conserved functional regions, indicating the conserved evolution of the circadian clock among organisms. These genes were expressed in all tissues, with high expression in the labial palps and hepatopancreas, suggesting a complex peripheral biological clock in S. constricta and the critical role of these two tissues in circadian functions. When exposed to different light regimes in labial palps, genes from the positive loop (ScClock and ScBmal) displayed a daily/circadian rhythm under both LD and DD and their acrophases were shifted by 5–7 h, whereas genes from the negative loop displayed daily rhythms (ScCry1 and ScPer) under LD and circadian rhythms (ScCry2 and ScTimeless2) under DD. However, only one gene (ScTimeless2) displayed a circadian rhythm under LL conditions. These results suggest that the S. constricta circadian system may be regulated by different clock genes to adapt to various light environments. Although similar phenomena were observed in the hepatopancreas, markedly different clock gene expression patterns occurred between the two tissues, indicating a tissue-specific regulated circadian system. Collectively, this study reveals how the circadian oscillators of S. constricta respond to light and may promote light environment optimization in the S. constricta farming industry. • The molecular circadian clock was conserved in S. constricta. • A peripheral and tissue-specific clock might exist in S. constricta. • The S. constricta circadian clock was synchronized by photoperiod. • A functional clock exists in S. constricta. [ABSTRACT FROM AUTHOR]

Fatty acid and sterol changes in razor clam Sinonovacula constricta (Lamarck 1818) reared at different salinities were investigated. In one experiment, juvenile S. constricta with shell length of 1.63 ± 0.11 mm were firstly cultured at 13 psu for 10 days, and then cultured at 8, 13, 18, and 23 psu for another 5 days. In another experiment, adult S. constricta with shell length of 15.54 ± 0.80 mm were cultured at 13 and 23 psu for 40 days. On the 25th day, half of the S. constricta cultured at 13 psu were transferred to 23 psu for another 15 days. The results showed that higher salinity significantly decreased growth rate and crude lipid content in S. constricta . The proportions of long chain-polyunsaturated fatty acids (LC-PUFAs, C ≥ 20, double bonds ≥ 3) were significantly increased in S. constricta cultured at higher salinity as compared with those at lower salinity. The proportion of cholesterol was firstly increased and then significantly decreased with increasing salinity. The proportion of brassicasterol was higher in S. constricta cultured at higher salinity, while proportions of other plant sterols showed no obvious change trends. Interestingly, when S. constricta were around 60 days old with shell length of 32.06 ± 1.13 mm, the inhibitory effect of high culture salinity on growth rate was weakened, but the nutritional value was significantly improved. The results of this study suggest an optimal culture mode for S. constricta , i.e. if S. constricta were firstly cultured at lower salinity (10–15 psu) and then exposed to higher salinity (20–25 psu) for a short period of time before harvest, a combination of both optimal production and nutritional value would be achieved. [ABSTRACT FROM AUTHOR]

In the present study, we aimed to explore the optimal light intensity for the breeding of Sinonovacula constricta , and the effect of light intensity on the growth of S. constricta at different developmental stages was studied. Four developmental stages (spawning, fertilized eggs, planktonic larvae, and juveniles) and different light intensities were set up in our current experiments. The results showed that various light intensities had significantly different effects on spawning amount and initial spawning time, while it had little influence on the hatchability of fertilized eggs (P > 0.05). The spawning amount of the razor clam peaked at a light intensity of 44–250 lx (P < 0.05). In the planktonic larvae experiment, the highest survival rate appeared at 487 ± 31 lx (58.61 ± 4.0%).For juvenile S. constricta , two substratum thicknesses (1 and 5 mm) and four light intensities, including L 1 (14 ± 11 lx), L 2 (380 ± 145 lx), L 3 (3792 ± 2105 lx), and L 4 (12,244 ± 5290 lx), were set up in a 10-day experiment. When the substratum thickness of 1 mm was applied, the body length and survival rate of S. constricta juveniles (0.26 ± 0.02 mm) in the L 2 group were found to be the largest, which were significantly different compared with the other groups. When the substratum thickness was changed to 5 mm, the body length and survival rate among those groups showed no significant difference. However, when the shellfish samples were changed to a larger size of razor clam (1.45 ± 0.16 mm), a more obvious effect of light intensity on the growth was observed in a 1-mm substratum group compared with the 5-mm group. These results indicated that light intensity exerted a significant effect on the growth of S. constricta at different stages, and each stage exhibited a distinct sensitivity to light intensity. Further study from the aspects of photoreceptors and relevant genes is currently in progress in our laboratory. • Spawning, hatching and growth of S. constricta were affected by light intensity. • All developmental stages of S. constricta were photosensitive, except zygote. • Optimal light intensity is critical for S. constricta breeding and farming. [ABSTRACT FROM AUTHOR]

Sinonovacula constricta is the first marine mollusc demonstrated to possess all fatty acyl desaturase (Fad) and elongase of very long chain fatty acids (Elovl) activities required for long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis via the Sprecher pathway, providing a good opportunity to investigate the essential fatty acids (FA) for its larval development. In the present study, we analyzed the Fad and Elovl expressions, FA compositions, and the feed effects of three representative microalgae (Chlorella sp. , Chaetoceros calcitrans and Isochrysis galbana) in S. constricta at early developmental stages. The results showed that S. constricta Fad and Elovl were all highly expressed in zygotes, which might occur from maternal transfer. Compared to transcripts of Elovl4 activity, Elovl2/5 was only expressed relatively high in zygotes, indicating that C ≥ 22 LC-PUFA were more important than C ≤ 20 LC-PUFA for S. constricta larval development. The zygotes accumulated abundant FA from parents, especially eicosapentaenoic acid (EPA), but with small amount of docosahexaenoic acid (DHA). Meanwhile, the trochophore larva fed with single or mixed microalgae with I. galbana (rich in DHA) exhibited significantly higher growth and survival rate compared to those fed with single Chlorella sp. (rich in 18:2n-6 and 18:3n-3) and C. calcitrans (rich in EPA) or both of them. Those findings suggested that the highly expressed Fad and Elovl might be largely responsible for DHA biosynthesis, but this capability was limited to meet the developmental requirement and the dietary DHA was necessary. Taken together, our results compellingly demonstrated that DHA was essential for S. constricta larval development, which is important to guide its breeding. • Fad and Elovl expressions were examined in developing S. constricta. • Fatty acid compositions were analyzed in developing S. constricta. • Trochophore larval growth and survival were evaluated by first-feeding diet. • DHA still appeared to be essential for S. constricta larval development. • Microalgae rich in EPA and DHA were critical for S. constricta breeding. [ABSTRACT FROM AUTHOR]