Genome-Wide Characterization and Analysis of Expression of the Histone Gene Family in Razor Clam, Sinonovacula constricta

The Chinese razor clam (Sinonovacula constricta), a bivalve species widely distributed in estuaries and mudflats, is often exposed to extreme environmental and microbial stresses. Histones are fundamental components of chromatin and play an important role in innate immunity, as demonstrated by its antimicrobial activities in clams. However, little attention has been paid to histones in bivalves. To fill this gap, we investigated the genomic distribution, structural characteristics, conserved motifs, and phylogenetic relationships of histones in S. constricta. A total of 114 histone genes were detected in the S. constricta genome, which were divided into 25 types in phylogenetic analysis. Among them, partial histones exhibited a tissue-dependent expression pattern, indicating that they may be involved in sustaining the homeostasis of organs/tissues in adult S. constricta. Furthermore, mRNA expression of certain histones changed significantly in S. constricta when infected with Vibrio parahaemolyticus, suggesting that histones play a role in the immune defense of S. constricta. All together, this study on histone genes in S. constricta not only greatly expands our knowledge of histone function in the clam, but also histone evolution in molluscs.

Characterizing the Role of Glutamine synthetase Gene on Ammonia Nitrogen Detoxification Metabolism of the Razor Clam Sinonovacula constricta

Ammonia nitrogen is a common toxic substance in the aquatic system, which seriously threatens the survival and growth of clams. However, less is known about the ammonia metabolism and detoxification strategy in razor clam. In this study, the polymorphism of the Glutamine synthetase gene from Sinonovacula constricta (Sc-GS) was found to be related to ammonia tolerance. By comparing the coding sequence (CDS) region of Sc-GS from two geographical populations, a total of 14 and 12 single nucleotide polymorphisms (SNPs) were identified, respectively, of which 10 loci were shared between the two populations. Among them, the locus c.1133T > G exhibited an extremely significant and strong association with ammonia tolerance in both populations (P < 0.01), and it was missense mutation, which led to the amino acid change from leucine (Leu) to arginine (Arg). Furthermore, the results about H&E staining and immunohistochemistry of Sc-GS protein in gills and hepatopancreas revealed that it was specifically localized in the lateral cilia of gill filaments and the endothelial cells of hepatocytes. After inhibiting the Sc-GS expression by RNA interference (RNAi) technology, the transcript levels of Sc-GS were extremely significantly downregulated at 24, 48, 72, and 96 h (P < 0.01) in the hepatopancreas. Taken together, these results indicated that the Sc-GS gene may participate in ammonia metabolism. In addition, these results will help to demonstrate the role of Sc-GS in ammonia nitrogen metabolism and provide markers related to ammonia nitrogen tolerance for molecular marker-assisted selection (MAS) of the razor clam.

Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications

The complete mitochondrial genome of Cultellus attenuates, a new aquaculture species, was sequenced and compared with mitogenomes from seven species of Heterodonta bivalve mollusk in GenBank. The mitochondrial genome of C. attenuatus is 16888 bp in length and contains 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs, and all genes are encoded on the same strand. In comparison with C. attenuates, the mitochondrial genes of Sinonovacula constricta from the same family were not rearranged, but those of six other species from different families were rearranged to different degrees. The largest noncoding region of C. attenuatus is 1173 bp in length and has an A + T content of 68.24%, located between nad2 and trnK. The results of phylogenetic analysis show that C. attenuates and S. constricta belonging to Cultellidae cluster into one branch while two species of Solenidae (Solen grandis and Solen strictus) cluster as their sister taxa. In conclusion, we used the mitochondrial genome data to demonstrate the closest relationship between C. attenuatus and S. constricta in Heterodonta. These data not only contribute to the understanding of the phylogenetic relationship of Heterodonta but also serve as a resource for the development of genetic markers in aquaculture.

The Discovery of Circadian Rhythm of Feeding Time on Digestive Enzymes Activity and Their Gene Expression in Sinonovacula constricta Within a Light/Dark Cycle

The circadian rhythm has a great impact on the growth, metabolism and development of animals, but little is known about the circadian rhythm of marine bivalves. Understanding of the feeding rhythm is of great significance to increase the yield of razor clam Sinonovacula constricta, an economically important bivalve mollusk. The aim of this experiment was to study the effects of circadian rhythm of feeding time on digestive enzymes activities and their gene expression in S. constricta within a light (ZT8-ZT20)/dark (ZT20-ZT8) cycle. The present results showed that circadian rhythm of feeding rate (FR) was highly associated with digestive enzyme activities and relative expression of their genes. The highest values of FR were basically observed in the night from ZT0-ZT2 and ZT6-ZT8, which were significantly higher than those values in the daytime from ZT12-ZT14 and ZT18-ZT20 (P < 0.05). The digestive enzymes activities displayed the highest values at ZT2 and ZT8, and the lowest at ZT14 and ZT20. Among them, cellulase and pepsin were found to have significantly different activities (P < 0.05), rather than amylase and lipase. Notably, the relative expression of digestive enzyme genes shared the similar pattern with the activities of digestive enzymes. The highest values of relative gene expression of amylase (AMY), lipase (LIP), cellulase (CEL), and pepsin (PEP) were found at ZT2 and ZT8 in the night, while the lowest values were found at ZT14 during the day. It is therefore suggested that the biological clock may regulate the process from feeding to digestion. Furthermore, it might be better to feed at night to reduce cultivating cost and increase economic benefits in the farming industry of S. constricta.

Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications

The complete mitochondrial genome of Cultellus attenuates, a new aquaculture species, was sequenced and compared with mitogenomes from seven species of Heterodonta bivalve mollusk in the gene bank. The mitochondrial genome of C. attenuatus is 16888bp in length and contains 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs, and all genes are encoded on the same strand. In comparison with C. attenuates, the mitochondrial genes of the Sinonovacula constricta from the same family were not rearranged, but those of six other species from different family were rearranged to different degrees. The largest non-coding region of C. attenuatus is 1173bp in length and with the A + T content of 68.24%, located between nad2 and trnK. The results of phylogenetic analysis show that the C. attenuates and the S. constricta belonging to Cultellidae cluster into one branch while two species of Solenidae ( Solen grandis and Solen strictus) are clustering as their sister taxon. These data not only contribute to the understanding of the phylogenetic relationship of the Heterodonta, but also serve as a resource for the development of the genetic markers in aquaculture.

Gonad Transcriptome Analysis of the Razor Clam (Sinonovacula constricta) Revealed Potential Sex-Related Genes

The razor clam, Sinonovacula constricta is a commercially important bivalve in the western Pacific Ocean, yet little is known about the mechanisms of sex determination/differentiation and gametogenesis. In the present study, the comparative transcriptome analysis of adult gonads (female gonads and male gonads) was conducted to identify potential sex-related genes in S. constricta. The number of reads generated for each target library (three females and three males) ranged from 31,853,422 to 37,750,848, and 20,489,472 to 26,152,448 could be mapped to the reference genome of S. constricta (the map percentage ranging from 63.71 to 71.48%). A total of 8,497 genes were identified to be differentially expressed between the female and male gonads, of which 4,253 were female-biased (upregulated in females), and 4,244 were male-biased. Forty-five genes were identified as potential sex-related genes, including DmrtA2, Sox9, Fem-1b, and Fem-1c involved in sex determination/differentiation and Vg, CYP17A1, SOHLH2, and TSSK involved in gametogenesis. The expression profiles of 12 genes were validated by qRT-PCR, which further confirmed the reliability and accuracy of the RNA-Seq results. Our results provide basic information about the genes involved in sex determination/differentiation and gametogenesis, and pave the way for further studies on reproduction and breeding in S. constricta and other marine bivalves.