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.

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.

Effects of dietary supplementation of three strains of Lactococcus lactis on HIFs genes family expression of the common carp following Aeromonas hydrophila infection.

HIFs (Hypoxia inducible factors) are the main regulators of the expression change of oxygen-dependent genes, in addition, they also play important roles in immune regulation. HIFs participate in infectious diseases and inflammatory responses, providing us a new therapeutic target for the treatment of diseases. In this study, 16 HIFs were identified in common carp genome database. Comparative genomics analysis showed large expansion of HIF gene family and approved the four round whole genome duplication (WGD) event in common carp. To further understand the function of HIFs, the domain architectures were predicted. All HIF proteins had the conserved HLH-PAS domain, which were essential for them to form dimer and bind to the downstream targets. The differences in domain of HIFα and HIFß might result in their different functions. Phylogenetic analysis revealed that all HIFs were divided into two subfamilies and the HIFs in common carp were clustered with their teleost counterparts indicating they are highly conservative during evolution. In addition, the tissue distribution was examined by RT-PCR showed that most of HIF genes had a wide range of tissue distribution but exhibited tissue-specific expression patterns. The expression divergences were observed between the copy genes, for example, HIF1A-1, HIF2A-1, ARNT-2 had wide tissue distribution while their copies had limited tissue distribution, proving the function divergence of copies post the WGD event. In order to find an effective activation of HIFs and apply to treatment of aquatic diseases, we investigate the dietary supplementation effects of different strains of Lactococcus lactis on the expression of HIFα subfamily members in kidney of common carp infected with A. hydrophila. In addition, all of the HIF genes have a high expression in the early stages of infection, and decreased in the treatment time point of 48 h in common carp. This phenomenon confirms that as a switch, the main function of HIFs is to regulate the production of immune response factors in early infection. So activation of the switch may be an effective method for infectious disease treatment. As expected, the treatment groups improved the expression of HIFs compared with the control group, and the effects of the three strains are different. The strain1 of L. lactis had a stronger induction on HIF genes than strain2 and strain3, and it might be applied as a potential activation of HIF genes for disease treatment. So, adding befitting L. lactis maybe a well method to activate the HIF genes to protect them from mycobacterial infection.