Sinonovacula Constricta Research Articles

Characterization of Vibrio mediterranei Isolates as Causative Agents of Vibriosis in Marine Bivalves.

Marine bivalves include species important globally for aquaculture and estuary ecology. However, epizootics of variable etiologies often pose a threat to the marine fishery industry and ecosystem by causing significant mortalities in related species. One of such diseases is larval vibriosis caused by bacteria of the genus Vibrio, which frequently occurs and causes mass mortalities in bivalve larvae and juveniles in hatcheries. During a mass mortality of razor clam, Sinonovacula constricta, juveniles in a shellfish hatchery in 2019, Vibrio mediterranei was identified as a dominant bacterial species in diseased animals and their rearing water. In this study, we selected and characterized 11 V. mediterranei isolates and studied their pathogenicity to the larvae and juveniles of S. constricta and Crossostrea sikamea. We found that V. mediterranei isolates showed various degrees of pathogenicity to the experimental animals by immersion. Injection of the extracellular products (ECPs) of the strains into clam juveniles resulted in similar pathogenicity with strain immersion. Furthermore, the measurements of enzyme activity suggested the existence of virulence factors in the ECPs of disease-causing V. mediterranei strains. Additionally, proteomic analysis revealed that more than 700 differentially expressed proteins were detected in the ECPs among V. mediterranei strains with different levels of virulence, and the higher expressed proteins in the ECPs of highly virulent strains were involved mainly in the virulence-related pathways. This research represented the first characterization of the V. mediterranei strains as causative agents for larval bivalve vibriosis. The mechanisms underlying the pathogenicity and related strain variability are under further study. IMPORTANCE In the marine environment, Vibrio members have a significant impact on aquatic organisms. Larval vibriosis, caused by bacteria of the genus Vibrio, often poses a threat to the marine fishery industry and ecosystem by causing the mortality of bivalves. However, the emerging pathogens of larval vibriosis in bivalves have not been explored fully. Vibrio mediterranei, the dominant bacterium isolated from moribund clam juveniles in a mortality event, may be responsible for the massive mortality of bivalve juveniles and vibriosis occurrence. Thus, it is necessary to study the pathogenic mechanisms of V. mediterranei to bivalve larvae. We found that V. mediterranei was the pathogen of larval bivalve vibriosis, and its extracellular products contributed a critical role for virulence in juveniles. This research is the first report of V. mediterranei as a causative agent for vibriosis in bivalve juveniles. Our results provide valuable information for understanding the pathogenic mechanism of V. mediterranei to bivalve larvae.

Effects of light on growth, feeding rate, digestion, and antioxidation in juvenile razor clams Sinonovacula constricta

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.

Comprehensive Analysis of Whole-Transcriptome Profiles in Response to Acute Hypersaline Challenge in Chinese Razor Clam Sinonovacula constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Chromosome-level genome assembly of the Pacific geoduck Panopea generosa reveals major inter- and intrachromosomal rearrangements and substantial expansion of the copine gene family.

The Pacific geoduck Panopea generosa (class Bivalvia, order Adapedonta, family Hiatellidae, genus Panopea) is the largest known burrowing bivalve with considerable commercial value. Pacific geoduck and other geoduck clams play important roles in maintaining ecosystem health for their filter feeding habit and coupling pelagic and benthic processes. Here, we report a high-quality chromosome-level genome assembly of P. generosa to characterize its phylogeny and molecular mechanisms of its life strategies. The assembled P. generosa genome consists of 19 chromosomes with a size of 1.47 Gb, a contig N50 length of 1.6 Mb, and a scaffold N50 length of 73.8 Mb. The BUSCO test of the genome assembly showed 93.0% completeness. Constructed chromosome synteny revealed many occurrences of inter- and intrachromosomal rearrangements between P. generosa and Sinonovacula constricta. Of the 35,034 predicted protein-coding genes, 30,700 (87.6%) could be functionally annotated in public databases, indicating the high quality of genome annotation. Comparison of gene copy numbers of gene families among P. generosa and 11 selected species identified 507 rapidly expanded P. generosa gene families that are functionally enriched in immune and gonad development and may be involved in its complex survival strategies. In particular, genes carrying the copine domains underwent additional duplications in P. generosa, which might be important for neuronal development and immune response. The availability of a fully annotated chromosome-level genome provides a valuable dataset for genetic breeding of P. generosa.

Genome-wide characterization of the cytosolic sulfotransferase 1B member 1 (SULT1B1) family and its expression responses to sulfide stress in the razor clam Sinonovacula constricta

The razor clam (Sinonovacula constricta), a typical burrowing organism in the intertidal zones, is often exposed to sulfide environment and shows strong sulfide tolerance. Located downstream of the sulfur metabolism pathway, cytosolic sulfotransferase family 1B member 1 (SULT1B1) is a key enzyme catalysing the sulfonation reaction, and plays an important role in the biotransformation of endogenous substances such as thyroid hormones (THs). To investigate their roles in sulfide resistance, a systematic analysis of S. constricta SULT1B1s (ScSULT1B1s), including genomic distribution, phylogenetic relationships, gene structure, conserved motifs, and expression profiles under sulfide stress, was performed. A total of 10 ScSULT1B1 genes were found in the S. constricta genome. Sequence analysis showed that ScSULT1B1 gene family encoded 155–425 amino acids, containing four catalytic active sites (K, N, H, and S), one PAPS binding domain at the N-terminus, and one PAPS binding and dimerization domain at the C-terminus. The spatial–temporal expression patterns of ScSULT1B1s were further estimated by quantitative real-time PCR (qRT-PCR). Among them, partial ScSULT1B1s showed significantly high expression in the gill, hepatopancreas, and siphon. Furthermore, the response expression of certain ScSULT1B1s significantly fluctuated under sulfide stress. Together, our results suggest that ScSULT1B1s, by mediating the sulfonation reaction, may regulate THs levels to maintain basic metabolic and immune functions, making S. constricta highly sulfide tolerant.

Circadian clock genes Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion in razor clam Sinonovacula constricta

The circadian clock is an endogenous regulation mechanism that coordinates biological processes with daily changes, which are regulated by circadian clock genes. Bmal1 and Period are key circadian clock genes and their roles in reproductive development have been widely studied. The spawning time of Sinonovacula constricta is limited to the night even under external artificial stimulation, and it might be regulated by the internal circadian clock. In this study, the heart rate of S. constricta was higher between 20:00-04:00 at night and lower between 12:00-16:00 during the day, and the sex hormone contents were the highest at 00:00 and the lowest at 18:00 (P < 0.01). Therefore, these obvious changes in the circadian rhythm indicate that S. constricta is a nocturnal animal. The open reading frame (ORF) of Bmal1 comprises 1944 bp encoding 647 aa, while the ORF of Period comprises 3111 bp encoding 1036 aa. Bmal1 and Period were both expressed in four tissues, but they had opposite rhythmic expression patterns. Bmal1 expression was higher at 00:00-06:00 and lower at 12:00-18:00, and Period expression was opposite, thereby suggesting that Bmal1 and Period are involved in positive and negative pathways regulated by the circadian clock, respectively. Strong protein fluorescence signals of Bmal1 and Period proteins were observed in mature oocytes, spermatids, hepatocytes, and epithelial cells of siphons. After siRNA interference, the expression of both Bmal1 and Period significantly decreased (P < 0.01), and the sex hormone contents decreased significantly from 3 to 7 days in the siRNA treatment groups (P < 0.01). Therefore Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion. These findings provide a theoretical basis for understanding the molecular mechanism related to spawning, and may facilitate the artificial propagation of mollusks.

Light entrainment and molecular regulation of the circadian clock in razor clam Sinonovacula constricta

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.

Optimal Culture Capacity of White Shrimp (Litopenaeus vannamei) and Razor Clam (Sinonovacula constricta) in a New Series-Connection Culture Model

Optimal Culture Capacity of White Shrimp (Litopenaeus vannamei) and Razor Clam (Sinonovacula constricta) in a New Series-Connection Culture Model

Physiological response of the razor clam Sinonovacula constricta exposed to hyposalinity stress

The razor clam Sinonovacula constricta is an economically important bivalve species in China, which inhabits the mudflat of intertidal and estuaries areas and frequently suffers the low salinity stress due to heavy rain events. The present study investigated the effects of hyposalinity stress (salinity 10, 5, 3, and 1) on osmoregulation and haemolymph microbiota in S. constricta after 7 days of exposure. The results showed that the survival rate of razor clams was only significantly reduced in the 1 ppt group compared to the control group (salinity 20) (P < 0.05). Haemolymph osmolality in razor clams reduced significantly with decreasing salinities (P < 0.05). Taurine, arginine, alanine and ornithine were the dominant free amino acids (FAAs) with higher contents in the haemolymph. Exposure to low salinities altered the composition of haemolymph FAAs, with alanine being the highest in the control group and taurine being the highest in the low salinity (LS) groups. Hyposalinity stress caused an increase in Na+/K+-ATPase (NKA) activity in gills, and histological analysis revealed severe hyperplasia in gills and a significant reduction in the length of gill filaments (P < 0.05). The microbial community variation in the haemolymph assessed by 16S rRNA sequencing explains that bacterial communities was shifted under low salinity stress. At the genus level, the relative abundance of Marinobacterium, Marinomonas and Pseudomonas changed significantly under hyposalinity stress (P < 0.05). These findings give further insight into the physiological responses and potential adaptation strategies of razor clams to hyposalinity stress and provide information for the development of inland low salinity culture.

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.

Diversity and distribution of Perkinsus spp. along the coast of China: Implications for widespread transmission of Perkinsus spp. in mollusks

Perkinsus species, which are parasitic pathogens of mollusks, have been transmitted and dispersed to various molluscan species along the coastal waters of many countries. However, few studies have addressed the diversity and distribution of Perkinsus spp. along the coast of China. Here we used conventional PCR amplification and sequencing techniques, combined with a qPCR assay as a confirmatory test, to evaluate the prevalence of Perkinsus species in molluscan species among different sea regions of China. Three Perkinsus species—P. olseni, P. beihaiensis, and P. chesapeaki—were detected, with P. chesapeaki reported for the first time along the Chinese coast. Seven of eight molluscan species carried Perkinsus species, including Crassostrea gigas, C. hongkongensis, Sinonovacula constricta, Ruditapes philippinarum, Scapharca subcrenata, Meretrix lyrate, and Haliotis diversicolor. Perkinsus olseni was prominent in the Yellow and Bohai Sea and East China Sea, while P. beihaiensis was prominent in the South China Sea. Most of the molluscan species carried Perkinsus spp. with the medium or low levels of PCR-prevalence (&amp;lt;30%). The three Perkinsus species possess high levels of internal transcriber spacer haplotypes, some of which are shared among many countries. The much higher PCR-prevalence of Perkinsus spp. in the clam Ruditapes philippinarum and the oyster Crassostrea hongkongensis suggests that Perkinsus species may be transmitted and dispersed to other mollusks through the transportation of Perkinsus-carried R. philippinarum and C. hongkongensis. Perkinsus carrying tended to be generally linked with a broader geographic range, lower prevalence, more diversified molluscan hosts, and more diversified Perkinsus haplotypes.

Benthic suspension-feeding clams affect sedimentary microbial communities and nitrogen cycling in seawater pond IMTA

Suspension-feeding bivalves are the key driver unit for cycling of material and energy in integrated multi-trophic aquaculture (IMTA) system, but only few studies have examined their bioturbation effects in land-based aquaculture system. In the present study we explored the effects of different species (Ruditapes philippinarum, Sinonovacula constricta and Mercenaria mercenaria) and stocking density of benthic clams on the structure and function (specifically, nitrification and denitrification) in pond IMTA using a medium-scale mesocosm incubation system. It was found that co-cultured with 3 benthic clam species respectively decreased the inorganic nitrogen and phosphate in water column, affected the sediment microbial communities, and increased abundance of nitrogen cycling related genes, such as AOB, nifH and nosZ, in sediment. Moreover, the moderate density of R. philippinarum (125 ind. /m2, weight of clam at 4.5 g ind.−1) have a higher activities of nitrogen cycling. The results are of great value on discovering the ecological mechanism of pond IMTA and improve the current IMTA models.

Impact of ocean acidification on the physiology of digestive gland of razor clams Sinonovacula constricta

Ocean acidification (OA) can have widespread implications for marine bivalves. While our current understanding of OA effects on the physiological performance is increasing, very little is known about the physiology of digestive gland of marine bivalves in response to OA. Here, we examined how the digestive system of razor clams (Sinonovacula constricta) responded to OA. Following 35-day exposure to CO2-driven seawater acidification, no significant decreases in phenotypic traits, such as dry body weight gain, specific growth rate, condition index and survival rate, as well physiological functions, such as activities of antioxidant and digestive enzymes, were observed, demonstrating the resistance of razor clams under acidified conditions. Histological results showed that some direct damages on the structure of digestive gland was observed, including degradation of digestive tubular, atrophy of epithelial cells, loose cell arrangement, even diffuse. This study provides insights into the digestive performance of marine bivalves in a rapidly acidifying ocean.

Massive expansion of P-selectin genes in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria: evidence from comparative genomics of Bivalvia

BackgroundP-selectin is a molecule participating in the inflammatory response through mediating cellular adhesion and essential for wound repair. However, studies regarding P-selectin in Bivalvia are rare. This study identified 90 P-selectin genes among nine bivalve genomes and classified them into 4 subfamilies according to phylogenetic analysis.ResultsNotable P-selectin gene expansion was observed in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria. The synteny analysis revealed that P-selectin gene expansion was mostly caused by tandem duplication. In addition, the expression profiles of P-selectin genes in S. constricta showed that many P-selectins were specifically highly expressed in the gills, and the P-selectin expression patterns changed dramatically under low salt stress and ammonia nitrogen stress.ConclusionsThe massive expansion of P-selectins may facilitate the tolerance to environmental stresses. This study sheds light on the characterizations and expression profiles of P-selectin genes in Bivalvia and provides an integrated framework for further investigation of the role of P-selectins in the environmental tolerance of bivalves.

Effect of Sinonovacula constricta on sediment microbial numbers and easily degradable organics in shrimp-crab polyculture systems

To explore the influence of different densities of Sinonovacula constricta on the composition of easily degradable organic matter and related functional bacteria, four experimental ecosystems were established: three polyculture systems (PMB1, PMB2, and PMB3) of Portunus trituberculatus and Marsupenaeus japonicus with different stocking densities of S. constricta (11.6, 23.1, and 34.7×104 ind./hm2, respectively) and a polyculture system with only P. trituberculatus and M. japonicus (PM). Among the easily degradable organic components in all aquaculture systems, protein content was the highest (0.74%~0.86%), followed by carbohydrates (0.16%~0.21%) and lipids (0.06%~0.13%). In the high-density (34.7×104 ind./hm2) S. constricta mixed culture system, the contents of carbohydrates, lipids, and proteins in the sediment were significantly lower than those of the other polyculture systems. The number of cellulose-decomposing bacteria in PMB3 was 3.79×106 cfu/g, which was significantly higher than that in the other systems. The number of starch-degrading bacteria and glutin-degrading bacteria was the lowest in PMB3, 1.26×104 cfu/g, and 160.00 cfu/g, respectively. The number of lipid-degrading bacteria in PMB3 was 0.77×104 cfu/g, which was significantly lower than that in the other systems. The easily degradable organics content in sediment was significantly positively correlated with the corresponding functional bacteria. The results showed that mixed culture of S. constricta could reduce the content of easily degradable organics in the sediment of mariculture ponds and change the number of functional bacteria in the sediment and the availability of degradable organic sediments may determine the abundance of corresponding degradable bacteria.

Identification, Phylogeny and Expressional Profiles of Peptidoglycan Recognition Protein (PGRP) Gene Family in Sinonovacula constricta

Identification, Phylogeny and Expressional Profiles of Peptidoglycan Recognition Protein (PGRP) Gene Family in Sinonovacula constricta

Bacterial community in Sinonovacula constricta intestine and its relationship with culture environment

Although the importance of intestinal microbes to aquaculture animals has been recognized, the intestinal bacteria of Sinonovacula constricta and its culture environment are rarely studied. In this study, high-throughput sequencing was used to explore the intestinal bacterial communities of pond water, sediment, and S. constricta intestine. Significance analysis and principal coordinates analysis (PCoA) showed that there were significant differences in bacterial communities among animals' intestine, pond water, and sediment (p < 0.05). Venn analysis showed that intestinal bacteria shared a considerable number of OTUs (operational taxonomic units) with the sediment and water. SourceTracker analysis suggested that the contribution of sediment to the intestinal bacteria of S. constricta was much larger than that of rearing water. The Kruskal-Wallis test showed that the dominant bacterial taxa differed significantly between animals' intestines and the pond environment, and each of them has a unique bacterial composition. A network diagram indicated the complex positive and negative interactions between intestinal bacteria at the OTU level. Furthermore, BugBase analysis indicated that the bacterial contribution to potential pathogens in the animals' intestines is similar to that in sediments, suggesting that sediment was the main source of potential pathogens in S. constricta intestine. This study provided a theoretical basis for environmental regulation and disease prevention of S. constricta in aquaculture. KEY POINTS: • Culture environment had a significant effect on the intestinal bacterial community in S. constricta. • Sediment was a major source of intestinal bacteria and potentially pathogenic bacteria. • Complex positive and negative interactions existed between intestinal bacteria.

Melatonin in razor clam Sinonovacula constricta: Examination of metabolic pathways, tissue distribution, and daily rhythms

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.

Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications.

The mitochondrial genomes of three species in Solenoidea of Heterodonta have been reported, but the mitochondrial genes and phylogenetic relationships of Cultellus attenuatus, which also belongs to this superfamily and has high economic value, are unknown. The complete mitochondrial genome of C. attenuatus was sequenced and compared with mitogenomes of seven species of Heterodonta bivalve mollusks in GenBank. The mitochondrial genome of C. attenuatus has a length of 16,888bp and contains 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs. 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 location, size, and composition of the largest noncoding regions in eight species suggested a closer relationship between C. attenuatus and S. constricta. The phylogenetic analysis showed that C. attenuatus and S. constricta belonging to Cultellidae cluster into one branch and that two species of Solenidae (Solen grandis and Solen strictus) clustered as their sister taxa. Overall, we used mitochondrial genome data to demonstrate that C. attenuatus and S. constricta exhibit the closest relationship in Heterodonta. These data and analyses provide new insights into the phylogenetic relationships in Heterodonta.

Genome Wide Identification and Expression Profiling Indicate Expansion of Family I84 Protease Inhibitor via Gene Tandem Duplication and Divergence in Razor Clam Sinonovacula constricta.

Family I84 protease inhibitors represent a novel family in the MEROPS peptidase database and are likely unique for molluscan host defense. Two Family I84 members, scSI-1 and scSI-2, were reported from the razor clam Sinonovacula constricta in a previous research. In the present study, 12 additional genes, named scSI-3 to scSI-14, were identified via genome wide sequence analyses. Among them, 10 genes were predicted to have a signal sequence, but one (scSI-7) was not. Besides, one sequence (scSI-14) was likely to encode a prematurely terminated peptide. The predicted mature peptides shared characteristics including 12 conserved cysteine residues, isoelectric points of 4.98 to 6.11, and molecular weights of 7.1 to 9.3 kDa with previously reported family members. Four motifs were characterized in 13 predicted mature peptides (with exception of scSI-14), which shared two to four conserved cysteine residues, are possibly to form two functional domain comprised 6 cysteine residues, respectively. At genomic level, all the 14 razor clam Family I84 genes were organized into 3 exons and 2 introns; 13 of them clustered in 3 regions of 100 kb on 3 separate chromosomes, suggesting tandem duplications of related genes. The promoter region of all the 14 genes was predicted to share some transcription factor binding sites, in particular those responsive to pathological and physiological stimuli, but no shared motifs were identified. Analyses also revealed differences in expression patterns among the genes. One gene in a tandem duplicated gene pairs usually showed a higher expression level than the other whereas non-tandem duplicated genes exhibited a higher degree of correlation in expression level. In addition, 8 of the 14 genes demonstrated higher level of expression in Vibrio tolerant clams than in non-tolerant clams following challenges with Vibrio parahaemolyticus. These results generated important information about the evolution of Family I84 protease inhibitors in S. constricta.