Genome-Wide Identification of 5-HT Receptor Gene Family in Razor Clam Sinonovacula constricta and Their Circadian Rhythm Expression Analysis.

Serotonin (5-HT) is primarily distributed in the gastrointestinal and central nervous systems, where it plays a crucial role in regulating various physiological functions such as digestion, reproduction and establishing animal emotions. 5-HT is an effective oxytocin widely used in molluscan aquaculture, and its physiological functions are performed by binding to corresponding 5-HT receptors (5-HTRs). In this study, seven 5-HTR genes of Sinonovacula constricta (Sc5-HTRs) were identified and analyzed, and they were designated as Sc5-HT1A, Sc5-HT1D, Sc5-HT2-1, Sc5-HT2-2, Sc5-HT2-3, Sc5-HT4 and Sc5-HT6. Phylogenetic analysis showed that the seven Sc5-HTRs were conserved among mollusks, and the Sc5-HTRs were all transmembrane proteins. The seven Sc5-HTR genes were distributed on chromosome 1, 2, 13 and 14. After injecting 5-HT, there was a significant increase in mRNA expression levels of Sc5-HT1A (p < 0.05) and Sc5-HT2-3 (p < 0.01), while Sc5-HT4 decreased significantly (p < 0.01) compared to control groups which might be effective 5-HT receptors. Furthermore, two of the receptors (Sc5-HT2-3 and Sc5-HT4) were expressed in the circadian rhythm patterns, indicating their potential influence on the nocturnal spawning of S. constricta. Overall, these findings provide a theoretical basis for understanding the structures and functions of 5-HTR gene family members, and may facilitate the artificial propagation of mollusks.

Expression and Functional Analysis of AMT1 Gene Responding to High Ammonia Stress in Razor Clam (Sinonovacula constricta).

Ammonium transporter 1 (AMT1), a member of ammonia (NH3/NH4+) transport proteins, has been found to have ammonia transport activity in plants and microorganisms. However, the functional characteristics and molecular mechanisms of AMT1 in mollusks remain unclear. The razor clam (Sinonovacula constricta) is a suitable model species to explore the molecular mechanism of ammonia excretion because of the high concentration of ambient ammonia it is exposed to in the clam-fish-shrimp polyculture system. Here, the expression of AMT1 in S. constricta (Sc-AMT1) in response to high ammonia (12.85 mmol/L NH4Cl) stress was identified by real-time quantitative PCR (qRT-PCR), Western blotting, RNA interference, and immunofluorescence analysis. Additionally, the association between the SNP_g.15211125A > T linked with Sc-AMT1 and ammonia tolerance was validated by kompetitive allele-specific PCR (KASP). A significant upregulated expression of Sc-AMT1 was observed during ammonia exposure, and Sc-AMT1 was found to be localized in the flat cells of gill. Moreover, the interference with Sc-AMT1 significantly upregulated the hemolymph ammonia levels, accompanied by the increased mRNA expression of Rhesus glycoprotein (Rh). Taken together, our findings imply that AMT1 may be a primary contributor to ammonia excretion in S. constricta, which is the basis of their ability to inhabit benthic water with high ammonia levels.

PDIA6 involves the thermal stress response of razor clam, Sinonovacula constricta.

Protein disulfide isomerases A6 (PDIA6), an oxidoreductase and isomerase, catalyzes the oxidation reduction and isomerization of disulfide bonds, and serves as molecular chaperone to prevent the buildup of misfolded proteins under various environmental insults. However, the role of PDIA6 in mollusks remains largely obscure, although its multifunctional protein has been reported in other species under adverse conditions. To fill this gap, we identified PDIA6 from the razor clam Sinonovacula constricta (ScPDIA6) and investigated its expression patterns in response to thermal stress. Tissue distribution showed that the mRNA transcript of ScPDIA6 was ubiquitously expressed in nine tested tissues. Temporal expression profiles by qPCR revealed that ScPDIA6 in the gill and mantle was significantly increased by hyper-thermic treatment. Further, Western blot and immunofluorescence indicated that ScPDIA6 was significantly upregulated by thermal treatment at the protein level. Additionally, the survival test demonstrated that the viability of E. coli cells expressing recombinant ScPDIA6 protein increased at 42 °C compared with empty vector. Overall, these findings suggested that ScPDIA6 may play a pivotal role in counteracting thermal stress. This study will provide valuable reference data resource for understanding the potential role of PDIA6 in mollusks.

Characteristics of glutathione peroxidase gene and its responses to ammonia-N stress in razor clam Sinonovacula constricta.

Glutathione peroxidase (GPX) is a crucial enzyme in the antioxidant defense system. However, the previous studies on the structure and functions of mollusk GPX genes are still very limited. Here, we investigated the GPX gene from Sinonovacula constricta (Sc-GPX), and its expression profiles, protein localization, gene function and association with ammonia tolerance. The full length of sequence of Sc-GPX was 1781 bp, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Quantitative expression of seven adult tissues showed that Sc-GPX was most abundant in hepatopancreas, followed by gills. Furthermore, the enzyme activity of Sc-GPX in hepatopancreas increased significantly under different ammonia concentrations (100, 140, and 180 mg/L) (P < 0.01). Further, we explored the mRNA expression level, histological structure and histo-cellular localization in gills and hepatopancreas of Sc-GPX under 140 mg/L ammonia stress. The mRNA expression level in gills and hepatopancreas of Sc-GPX increased significantly (P < 0.05) and immunohistochemistry results suggested that the columnar cells of gills filaments and the endothelial cells of hepatopancreas were the major sites for the action of Sc-GPX protein. In addition, we performed western blotting (WB), RNA interference (RNAi) and single nucleotide polymorphisms (SNPs) in the hepatopancreas of Sc-GPX under ammonia stress (140 mg/L). WB results indicated that the protein expression of Sc-GPX increased significantly (P < 0.01) after ammonia challenge. In addition, expression of Sc-GPX mRNA were significantly downregulated at 24 and 48 h after RNAi (P < 0.01). The association analysis between ammonia-tolerance group and control group identified six SNPs in coding sequence (CDS) of Sc-GPX from 449 individuals. Among them, c.162A > C was missense mutation, which lead to the amino acid change from Lys to Asn. These findings revealed that Sc-GPX may play a critical role in clam ammonia detoxification.

Vital Role of Glutamate Dehydrogenase Gene in Ammonia Detoxification and the Association Between its SNPs and Ammonia Tolerance in Sinonovacula constricta.

Increasing evidence has revealed accumulated ammonia will cause adverse effects on the growth, reproduction, and survival of aquatic animals. As a marine benthic mollusk, the razor clam Sinonovacula constricta shows better growth and survival under high ammonia nitrogen environment. However, little is known about its adaptation mechanisms to high ammonia stress in an integrated mariculture system. In this study, we analyzed the association between the polymorphism of glutamate dehydrogenase gene (GDH), a key gene involved in ammonia nitrogen detoxification, and ammonia tolerance. The results showed that 26 and 22 single-nucleotide polymorphisms (SNPs) of GDH in S. constricta (denoted as Sc-GDH) were identified from two geographical populations, respectively. Among them, two SNPs (c.323T > C and c.620C > T) exhibited a significant and strong association with ammonia tolerance, suggesting that Sc-GDH gene could serve as a potential genetic marker for molecular marker-assisted selection to increase survival rate and production of S. constricta. To observe the histological morphology and explore the histocellular localization of Sc-GDH, by paraffin section and hematoxylin-eosin staining, the gills were divided into gill filament (contains columnar and flattened cells) and gill cilia, whereas hepatopancreas was made up of individual hepatocytes. The results of immunohistochemistry indicated that the columnar cells of gill filaments and the endothelial cells of hepatocytes were the major sites for Sc-GDH secretion. Under ammonia stress (180 mg/L), the expression levels of Sc-GDH were extremely significantly downregulated at 24, 48, 72, and 96 h (P < 0.01) after RNA interference. Thus, we can speculate that Sc-GDH gene may play an important role in the defense process against ammonia stress. Overall, these findings laid a foundation for further research on the adaptive mechanisms to ammonia-nitrogen tolerance for S. constricta.

Genomic Insights into the Origin and Evolution of Molluscan Red-Bloodedness in the Blood Clam Tegillarca granosa.

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB.

The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure.

High ammonia can inhibit the survival and growth, and even cause mortality of razor clam (S. constricta). The accumulation of ammonia to lethal concentrations in some invertebrates may be partially prevented by converting some of the ammonia into glutamine (Gln). Glutamine dehydrogenase (GDH) and glutamine synthetase (GS) have been widely implicated a central role in response to ammonia stress. However, the molecular and physiological response of GDH and GS to ammonia alterations has not yet been determined in clams. To investigate the possible participatory role of GDH and GS genes in altered ammonia conditions, we have cloned their gene sequences and examined the mRNA expression and western blotting under ammonia exposure in S. constricta (ScGDH and ScGS), and detected the levels of GS and GDH, and the content of glutamate (Glu) and Gln. The full-length cDNA of ScGDH was 3924 bp, with a 1629 bp open reading frame (ORF) encoding a 542 amino-acid polypeptide. The complete cDNA sequence for ScGS had 2739 bp with an ORF of 1110 bp encoding 369 amino acids. To investigate ammonia detoxification strategies, the clams were exposed to ammonia for 96 h at four different concentrations (0, 100, 140, and 180 mg/L). Exposure to ammonia resulted in a significant increase of glutamate concentration and Gln in the haemocytes. GDH activity, GDH relative mRNA and protein expression, GS activity, GS relative mRNA and protein expression increased significantly and showed a pronounced time and dosage interaction in the liver. The results suggested that the protective strategies of Gln formation existed in S. constricta, which could convert ammonia to non- or less toxic nitrogenous compounds on the exposure of ammonia. Glutamate content in the haemocytes increased significantly, which is to ensure sufficient Glu to meet the needs for GS to catalyze the conversion of ammonia to Gln. We proposed that the induction of Glu synthesis-related genes and the subsequent formation of the active protein occurred in preparation for the increased capacity of the body to convert ammonia, into Gln. The results of this study suggested that GDH and GS play an important role in the synthesis of Gln, emphasizing, the protective strategies of Gln formation in S. constricta convert ammonia to nontoxic or less toxic nitrogenous compounds upon exposure to ammonia.

A Potential Role of Bone Morphogenetic Protein 7 in Shell Formation and Growth in the Razor Clam Sinonovacula constricta.

Bone morphogenetic proteins (BMPs) not only play essential roles in bone development but also are involved in embryonic growth, organogenesis cell proliferation and differentiation. However, the previous studies on the functions of shellfish BMPs genes are still very limited. To better understand its molecular structure and biological function, BMP7 of the razor clam Sinonovacula constricta (Sc-BMP7) was cloned and characterized in this study. The full length of Sc-BMP7 is 2252 bp, including an open reading frame (ORF) of 1257 bp encoding 418 amino acids. The protein sequence included a signal peptide (1-32 aa), a prodomain (38-270 aa) and a TGF-ß domain (317-418 aa). The quantitative expression of eleven adult tissues showed that Sc-BMP7 was significantly higher expressed in the gill, foot, and mantle (P < 0.05), but lower in hemocytes and hepatopancreas. In the early development stages, low expression was detected in the stages of unfertilized mature eggs, fertilized eggs, 4-cell embryos, blastula, gastrulae, whereas it increased after the stage of trochophore and demonstrated the highest expression in umbo larvae (P < 0.01). In shell repair experiment, Sc-BMP7 showed increasing expression level after 12 h. The higher expression of Sc-BMP7 was detected while Ca2+ concentration was reduced in seawater. After inhibiting Sc-BMP7 expression using RNA interference (RNAi) technology, expression of Sc-BMP7 mRNA and protein were significantly down-regulated (P < 0.05) in the central zone of mantle (nacre formation related tissue) and the pallial zone of mantle (prismatic layer formation related tissue). Association analysis identified two shared SNPs in exon of Sc-BMP7 gene from 246 individuals of two groups. These results indicated that BMP7 might be involved in shell formation and growth. These results would contribute to clarify the role of Sc-BMP7 in the regulation of growth and shell formation, and provide growth-related markers for molecular marker assisted breeding of this species.

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta.

Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates and plays a critical role in protein metabolism and growth. In the present study, we investigated the full-length cDNA sequence of the LAP3 gene in Sinonovacula constricta (ScLAP3) using expressed sequence tags and rapid amplification of cDNA ends. The full-length ScLAP3 cDNA was 2885 bp, with a 1560 bp open reading frame encoding 519 amino acids. Sequence analysis revealed that ScLAP3 shared 70.9% identity with LAP3 from the blood clam Tegillarca granosa and 62.0-68.0% with other species. ScLAP3 was expressed in all six tested tissues, with significantly higher expression levels in the foot compared with mantle, adductor muscle, liver, gills, and siphon tissues in adults (P < 0.01). In the eight developmental stages, ScLAP3 expression gradually increased, with significantly higher levels in D-shaped larvae compared with other developmental stages (P < 0.01), suggesting that it may be involved in the formation of certain organs during early development. Association analysis identified three shared single nucleotide polymorphisms (SNPs), c.1073A > G, c.1139C > T and c.1154A > G in exons of ScLAP3 gene from 177 individuals of two groups, one selective strain and one wild population, which had significant effects on growth traits of S. constricta. The results provided candidate genetic markers to assist selective breeding of razor clams toward improved growth.

Genetic analysis assessed by microsatellites for a diallel mating design of two geographical stocks of the blood clam Tegillarca granosa.

To determine the potential for productive efficiency and genetic improvement in the blood clam Tegillarca granosa, four offspring populations (ZZ, ZK, KZ and KK) were produced from a diallel mating of two different geographical stocks (Z and K). The levels of genetic diversity and population structures of four populations were analyzed using 14 polymorphic microsatellites. The results showed that the mean observed heterozygosities (Ho) of reciprocal cross populations (ZK and KZ) was higher than those of pure populations(ZZ and KK). The largest values of genetic differentiation coefficient (Fst = 0.067) and Nei's unbiased genetic distance (Dc = 0.263) were between ZK and KZ, and the smallest (Fst = 0.020, Dc = 0.116) were between ZZ and KK, which revealed that the largest genetic divergence was between the two reciprocal cross populations and the smallest was between two pure populations. This study demonstrated that the reciprocal cross populations of T. granosa had an extensive genetic difference and improvement, which may be advantageous for future breeding studies.

Polymorphism of the multiple hemoglobins in blood clam Tegillarca granosa and its association with disease resistance to Vibrio parahaemolyticus.

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, but it can serve additional functions beyond the transport of oxygen. In this study, we identified polymorphism in the blood clam Tegillarca granosa Hb (Tg-Hb) genes and investigated the association of this polymorphism with resistance/susceptibility to Vibrio parahaemolyticus. Analysis of the 540 sequences revealed 28 SNPs in the coding region of three Tg-Hbs, corresponding to about one SNP per 48 bp. Three SNPS: HbIIA-E2-146, HbIIB-E2-23, HbIIB-E2-121 showed a significant association with resistance/susceptibility to V. parahaemolyticus (P < 0.05). To further demonstrate that three significant SNPs of Tg-Hbs is associated with resistance of clams to V. parahaemolyticus, SNPs were genotyped in V. parahaemolyticus resistant strain clams and the wild base population from which this strain was derived. The results indicated that the nonsynonymous mutation T allele at HbIIA-E2-146 and A allele at HbIIB-E2-23 are associated with V. parahaemolyticus resistance in the blood clam, and its association with disease resistance may be due to its cause changes in amino acid sequences to a functional polymorphism. Together with previous bacterial challenge study, these results provides direct evidence that variation at HbIIA-E2-146 and HbIIB-E2-23 are associated with disease resistance in the blood clam, and these two polymorphic loci could be potential gene markers for the future molecular selection of strains that are resistant to diseases caused by V. parahaemolyticus.