NADK-mediated proline synthesis enhances high-salinity tolerance in the razor clam.

Euryhaline organisms can accumulate organic osmolytes to maintain osmotic balance between their internal and external environments. Proline is a pivotal organic small molecule and plays an important role in osmoregulation that enables marine shellfish to tolerate high-salinity conditions. During high-salinity challenge, NAD kinase (NADK) is involved in de novo synthesis of NADP(H) in living organisms, which serves as a reducing agent for the biosynthetic reactions. However, the role of shellfish NADK in proline biosynthesis remains elusive. In this study, we show the modulation of NADK on proline synthesis in the razor clam (Sinonovacula constricta) in response to osmotic stress. Under acute hypersaline conditions, gill tissues exhibited a significant increase in the expression of ScNADK. To elucidate the role of ScNADK in proline biosynthesis, we performed dsRNA interference in the expression of ScNADK in gill tissues to assess proline content and the expression levels of key enzyme genes involved in proline biosynthesis. The results indicate that the knock-down of ScNADK led to a significant decrease in proline content (P<0.01), as well as the expression levels of two proline synthetase genes P5CS and P5CR involved in the glutamate pathway. Razor clams preferred to use ornithine as substrate for proline synthesis when the glutamate pathway is blocked. Exogenous administration of proline greatly improved cell viability and mitigated cell apoptosis in gills. In conclusion, our results demonstrate the important role of ScNADK in augmenting proline production under high-salinity stress, by which the razor clam is able to accommodate salinity variations in the ecological niche.

Ammonia-induced oxidative stress triggered apoptosis in the razor clam (Sinonovacula constricta).

As one of the most significant contaminants and stressors in aquaculture systems, ammonia adversely jeopardizes the health of aquatic animals. Ammonia exposure affects the development, metabolism, and survival of shellfish. However, the responses of the innate immune and antioxidant systems and apoptosis in shellfish under ammonia stress have rarely been reported. In this study, razor clams (Sinonovacula constricta) were exposed to different concentrations of non-ion ammonia (0.25 mg/L, 2.5 mg/L) for 72 h and then placed in ammonia-free seawater for 72 h for recovery. The immune responses induced by ammonia stress on razor clams were investigated by antioxidant enzyme activities and degree of apoptosis in digestive gland and gill tissues at different time points. The results showed that exposure to a high concentration of ammonia greatly disrupted the antioxidant system of the razor clam by exacerbating the accumulation of reactive oxygen species ( O 2 - , H2O2) and disordering the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), and the level of activity remained at a significantly high level after recovering for 72 h (P < 0.05). In addition, there were significant differences (P < 0.05) in the expression of key genes (Caspase 7, Cyt-c, Bcl-2, and Bax) in the mitochondrial apoptotic pathway in the digestive glands and gills of razor clams as a result of ammonia stress and were unable to return to normal levels after 72 h of recovery. TUNEL staining indicated that apoptosis was more pronounced in gills, showing a dose and time-dependent pattern. As to the results, ammonia exposure leads to the activation of innate immunity in razor clams, disrupts the antioxidant system, and activates the mitochondrial pathway of apoptosis. This is important for comprehending the mechanism underlying the aquatic toxicity resulting from ammonia in shellfish.

Effects of acute hypoxia and reoxygenation on histological structure, antioxidant response, and apoptosis in razor clam Sinonovacula constricta.

Hypoxia is one of the major environmental problems limiting the healthy development of intensive aquaculture. Marine benthic shellfish are encountering heightened problems related to hypoxic stress as a result of ongoing human activities and aquaculture operations. Razor clam Sinonovacula constricta, a commercially valuable shellfish, has not yet been reported in studies on physiological changes caused by hypoxia and reoxygenation. To understand the negative effects of hypoxia and reoxygenation on the clams, we set up two low-oxygen concentration groups (DO 2.0 mg/L and DO 0.5 mg/L) and assessed multiple aspects of oxidative damage to their hepatopancreas and gills. After the hypoxic stress, the two tissues of the razor clam suffered varying degrees of damage, including cell degeneration and disruption of mitochondrial cristae. After reoxygenation, the 2.0 mg/L group recovered substantially, but the clams in the 0.5 mg/L group still unrecovered. The activities of antioxidant enzymes (MDA, T-AOC, SOD, GPX, and CAT) in clams were considerably altered by acute hypoxia and reoxygenation. Briefly, there was a growing and then declining trend in MDA, T-AOC, and SOD activities in the hepatopancreas, whereas GPX and CAT activities showed the converse trend. In the hepatopancreas and gills, the level of anti-apoptotic gene Bcl-2 transcripts gradually decreased with the duration of hypoxia and increased following reoxygenation. However, changes in the transcript level of the pro-apoptotic gene Bax were in contrast to that of Bcl-2. The TUNEL assay revealed that hypoxia caused apoptosis. Furthermore, at DO 0.5 mg/L, the degree of apoptosis was more significant than at DO 2.0 mg/L, and hepatopancreatic apoptosis was more severe than gill apoptosis. Collectively, our findings imply that hypoxia induces oxidative stress, histological damage, and apoptosis in razor clams in a concentration-dependent and tissue-specific manner. These consequences serve as a reminder that prolonged recovery periods may be required for razor clams to fully recover from oxidative damage resulting from hypoxia-reoxygenation episodes.

The Inhibitory Effects of RNA-Interference-Mediated Guanylate Cyclase Knockdown on Larval Metamorphosis and Early Progeny Growth of Razor Clam.

Guanylate cyclase (GC, cGMPase) is a key enzyme in organisms, catalyzing the synthesis of cGMP from GTP, thus making cGMP work. cGMP plays a vital role in the regulation of cell and biological growth as a second messenger in signaling pathways. In this study, we screened and identified cGMPase from the razor clam Sinonovacula constricta, which encoded 1257 amino acids and was widely expressed in different tissues, especially the gill and liver. We also screened one double-stranded RNA (dsRNA), cGMPase, which was used to knockdown cGMPase at three larval metamorphosis development stages: trochophores-veliger larve, veliger larve-umbo larve, and umbo larve-creeping larvae. We showed that interference at these stages significantly inhibited larval metamorphosis and survival rates. cGMPase knockdown resulted in an average metamorphosis rate of 60% and an average mortality rate of 50% when compared with control clams. After 50 days, shell length and body weight were inhibited to 53% and 66%, respectively. Thus, cGMPase appeared to regulate metamorphosis development and growth in S. constricta. By examining the role of the key gene in the metamorphosis development of S. constricta larvae and the growth and development period, we can provide some data reference for studying the growth and development mechanism of shellfish, and the results provided basic information for the breeding of S. constricta.

Domain-Dependent Evolution Explains Functional Homology of Protostome and Deuterostome Complement C3-Like Proteins.

Complement proteins emerged early in evolution but outside the vertebrate clade they are poorly characterized. An evolutionary model of C3 family members revealed that in contrast to vertebrates the evolutionary trajectory of C3-like genes in cnidarian, protostomes and invertebrate deuterostomes was highly divergent due to independent lineage and species-specific duplications. The deduced C3-like and vertebrate C3, C4 and C5 proteins had low sequence conservation, but extraordinarily high structural conservation and 2-chain and 3-chain protein isoforms repeatedly emerged. Functional characterization of three C3-like isoforms in a bivalve representative revealed that in common with vertebrates complement proteins they were cleaved into two subunits, b and a, and the latter regulated inflammation-related genes, chemotaxis and phagocytosis. Changes within the thioester bond cleavage sites and the a-subunit protein (ANATO domain) explained the functional differentiation of bivalve C3-like. The emergence of domain-related functions early during evolution explains the overlapping functions of bivalve C3-like and vertebrate C3, C4 and C5, despite low sequence conservation and indicates that evolutionary pressure acted to conserve protein domain organization rather than the primary sequence.

Expression of p38MAPK and its regulation of apoptosis under high temperature stress in the razor clam Sinonovacula constricta.

p38MAPK is a key branch of the MAPK (mitogen-activated protein kinase) pathway that plays an important role in physiological processes such as apoptosis, cell proliferation and growth. In this experiment, we screened and identified one p38MAPK gene in the razor clam Sinonovacula constricta, which encoded 359 amino acids and was widely expressed in various adult tissues. After 24 h of high temperature stress at 34 °C, the transcript expression of p38MAPK showed significant changes in all tested tissues. In particular in the gill and hepatopancreas tissues, where the expression increased 1.81 and 7.83 times compared with the control group, respectively (P < 0.01). Furthermore, we examined the expression of the apoptosis suppressor gene Bcl-2 and pro-apoptosis gene Bax by knock-down of p38MAPK with dsRNA interference in the gill and hepatopancreas tissues. The obvious up-regulation expression of Bcl-2 and significant suppression of Bax were observed, respectively (P < 0.01). Moreover, the TUNEL staining technique was used to detect apoptosis before and after interference. The degree of apoptosis in the gill and hepatopancreas tissues was reduced after interference with p38MAPK, and the ROS content was significantly reduced (P < 0.01). The results suggested that p38MAPK had a regulatory role in the heat tolerance of razor clams.

Two fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity.

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes.

Integrated analysis of transcriptomic and metabolomic data to evaluate responses to hypersalinity stress in the gill of the razor clam (Sinonovacula constricta).

Salinity is an important ecological factor that affects physiological metabolism, survival, and distribution of marine organisms. Despite changes in the osmolarity and composition of the cytosol during salinity shifts, marine mollusks are able to maintain their metabolic function. The razor clam (Sinonovacula constricta) survives the wide range of salinity in the intertidal zone via changes in behavior and physiology. To explore the stress responses and mechanisms of salinity tolerance in razor clams, we collected transcriptomic and metabolomic data from a control group (salinity 20‰, S20) and a salinity-stress group (salinity 35‰, S35). The transcriptome data showed that genes related to the immune system, cytoskeleton remodeling, and signal transduction pathways dominated in the S35 group to counteract hypersalinity stress in the gill. The metabolomic analysis showed that 142 metabolites were significantly different between the S35 and S20 groups and that amino acid and carbohydrate metabolism were affected by hypersalinity stress. Levels of amino acids and energy substances, such as l-proline, isoleucine, and fructose, were higher in the gill of the S35 group. The combination of transcriptomic and metabolomic data indicated that metabolism of amino acids, carbohydrates, and lipids was enhanced in the gill during adaptation to high salinity. These results clarified the complex physiological processes involved in the response to hyperosmotic stress and maintenance of metabolism in the gill of razor clams. These findings provide a reference for further study of the biological responses of euryhaline shellfish to hyperosmotic stress and a molecular basis for the search for populations with high salinity tolerance.

RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams.

Dopamine beta-hydroxylase (DßH) plays an essential role in the synthesis of catecholamines (CA) in neuroendocrine networks. In the razor clam, Sinonovacula constricta a novel gene for DßH (ScDßH-α) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScDßH-α gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScDßH-α in adult clams using siRNA caused a reduction in the growth rate compared to control clams. Reduced growth was associated with strong down-regulation of gene transcripts for the growth-related factors, platelet derived growth factors A (PDGF-A) (P < 0.001) 24 h after ScDßH-α knock-down, vascular endothelial growth factor (VEGF1) (P < 0.001) and platelet derived growth factor B (PDGF-B-2) (P < 0.001) 24 h and 48 h after ScDßH-α knock-down and transforming growth factor beta (TGF-ß1) (P < 0.001) 48 h and 72 h after ScDßH-α knock-down. Taken together the results suggest that the novel ScDßH-α gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.

Integrated mRNA and Small RNA Sequencing Reveals Regulatory Expression of Larval Metamorphosis of the Razor Clam.

The razor clam, Sinonovacula constricta, is an important economic marine shellfish, and its larval development involves obvious morphological and physiological changes. MicroRNA plays a key role in the physiological changes of the organism through regulating targeted mRNA. This study performed miRNA-mRNA sequencing for eight different developmental stages of S. constricta using Illumina sequencing. A total of 2156 miRNAs were obtained, including 2069 known miRNAs and 87 novel miRNAs. In addition, target genes were predicted for key miRNAs differentially expressed between adjacent development samples by integrating the mRNA transcriptome. Further analysis revealed that the differentially expressed genes were enriched in complement activation, alternative pathways, translation, and negative regulation of monocyte molecular protein-1 production. KEGG pathway annotation showed significant enrichment in the regulation of the ribosome, phagosome, tuberculosis and fluid shear stress, and atherosclerosis. Ten mRNAs and ten miRNAs that are related to larval metamorphosis were identified using real-time PCR. Furthermore, the double luciferase experiment validated the negative regulatory relationship between miR-133 and peroxisome proliferator-activated receptor-γ (PPAR-γ). These results indicated that the target genes regulated by these differentially expressed miRNAs may play an important regulatory role in the metamorphosis development of S. constricta.

Dopamine beta-hydroxylase and its role in regulating the growth and larval metamorphosis in Sinonovacula constricta.

Dopamine beta-hydroxylase (DßH) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The DßH gene was identified from the razor clam Sinonovacula constricta and referred to as ScDßH. The ScDßH gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScDßH transcript expression was abundant in liver and hemolymph. During early development, ScDßH expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of DßH were screened. After challenge with DßH inhibitor, the larval metamorphosis and survival rates, and juvenile growth were obviously decreased. Under the siRNA stress, the larval metamorphosis and survival rates were also significantly decreased. Therefore, ScDßH may play an important regulating role in larval metamorphosis and juvenile growth.

A four-CRD C-type lectin from razor clam Sinonovacula constricta mediates agglutination and phagocytosis.

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-binding proteins that play crucial roles in invertebrate immunity. In this study, a novel C-type lectin gene (ScCTL-1) was identified in razor clam Sinonovacula constricta. The ScCTL-1 gene, consisting of four C-type carbohydrate recognition domains (CRDs) with an N-terminal signal peptide and a C-terminal transmembrane region. The gene is widely expressed in almost all tissues, with the highest expression in the hepatopancreas. To explore the functional characteristics of this structurally novel gene, tests of binding specificity, agglutinating activity, and phagocytic promoting activity were included in this study. Bacterial stimulation up-regulated ScCTL-1 expression in hemocytes. The binding activity of rScCTL-1 to bacteria was tested in vitro, and bacterial agglutination was observed under the same conditions. Ca2+ was essential for carbohydrate binding. Additionally, rScCTL-1 promoted the phagocytic activity of hemocytes to varying degrees against different bacteria, unlike the classical opsonin. These results suggest ScCTL-1 is a classical immune-related C-type lectin possessing unique immune-related properties.

Identification of a dopamine receptor in Sinonovacula constricta and its antioxidant responses.

The dopamine (DA) D2 receptor is a member of the G protein-coupled receptors of organisms and plays an important role in immune system regulation. The presence of DA receptors has been widely reported in vertebrates, but few studies have been conducted in shellfish. Here, we identified a novel DA-D2 receptor gene, ScDopR2-1, in the razor clam Sinonovacula constricta. ScDopR2-1 belongs to the family of G protein-coupled receptors, containing seven hydrophobic transmembrane domains, along with 16 predicted N-glycosylation sites and 69 phosphorylation sites. A longer third intracellular loop and a shorter C-terminus in ScDopR2-1 are characteristic features of D2 receptors. ScDopR2-1 is widely expressed in tissues from adult clams, showing high expression in siphon and foot tissues. Furthermore, in response to Vibrio anguillarum challenge, ScDopR2-1 expression levels are significantly increased in liver tissue. Moreover, changes in the activities of catalase (CAT) and superoxide dismutase (SOD) also indicate that the organism causes an immune response. In summary, the results indicate that ScDopR2-1 plays a pivotal role in antioxidant responses in S. constricta.

Complement factor B/C2 in molluscs regulates agglutination and illuminates evolution of the Bf/C2 family.

Complement factor B/C2 family (Bf/C2F) proteins are core complement system components in vertebrates that are absent in invertebrates and have been lost by numerous species, raising evolutionary questions. At least 3 duplication events have occurred from Cnidaria (ancestor) to mammals. Type II Bf/C2 genes appeared during separation of Proterostomia and Deuterostomes. The second event occurred during separation of vertebrates and invertebrates, yielding type II-2 Bf/C2. The third event occurred when jawed and jawless fish were separated, eventually producing Bf and C2 genes. Herein, we report the second mollusc Sinonovacula constricta Bf/C2-type gene (ScBf). ScBf is similar to Ruditapes decussatus Bf-like because both lack the first complement control protein module at the N terminus present in mammalian Bf/C2 proteins. Uniquely, the Ser protease (SP) module at the C terminus of ScBf is ∼50 aa longer than in other complement factor B/C2-type (Bf/C2T) proteins, and is Glu-rich. Bf/C2T proteins in molluscs lack the catalytic Ser in the SP module. Surprisingly, ScBf regulates rabbit erythrocyte agglutination, during which it is localized on the erythrocyte surface. Thus, ScBf may mediate the agglutination cascade and may be an upstream regulator of this process. Our findings provide new insight into the origin of the Bf/C2F.-Peng, M., Li, Z., Niu, D., Liu, X., Dong, Z., Li, J. Complement factor B/C2 in molluscs regulates agglutination and illuminates evolution of the Bf/C2 family.

Tolerance, Growth, and Physiological Responses of the Juvenile Razor Clam (Sinonovacula constricta) to Environmental Ca2+ and Mg2+ Concentrations.

To facilitate transplanting razor clam (Sinonovacula constricta) populations to inland saline-alkaline waters (ISWs), we evaluated the tolerance of juvenile S. constricta (JSC) to Ca2+ and Mg2+ concentrations, and determined the effects of these ions on JSC growth and physiological parameters. After 30 days stress, the tolerable ranges of JSC to Ca2+ and Mg2+ were determined to be 0.19 mmol⋅L-1-19.46 mmol⋅L-1 and 0 mmol⋅L-1-29.54 mmol⋅L-1, respectively. The concentrations of Ca2+ (less than 0.65 mmol⋅L-1 or more than 3.24 mmol⋅L-1) and Mg2+ (less than 0.37 mmol⋅L-1 or more than 14.17 mmol⋅L-1) significantly inhibit JSC growth. Physiological enzyme activity no significant response when the concentrations range of Ca2+ and Mg2+ are 0.93 mmol⋅L-1-6.49 mmol⋅L-1 and 0.37 mmol⋅L-1-14.77 mmol⋅L-1, respectively. For transplantation practice, these data indicate that only high concentrations of Ca2+ (3.24-6.825 mmol⋅L-1) and Mg2+ (14.77-33.69 mmol⋅L-1) in target inland saline-alkaline water had significantly impact on growth and physiological response. In addition, present study suggests that the increase in Ca2+ and Mg2+ ion concentrations caused by ocean acidification will not affect the survival, growth and physiology of S. constricta. Current research suggests that S. constricta can adapt to extreme changes in the marine environment (Ca2+ and Mg2+) and may be an excellent candidate for inland saline-alkaline water transplantation practice.

Post-transcriptional regulation through alternative splicing after infection with Flavobacterium columnare in channel catfish (Ictalurus punctatus).

Columnaris disease has long been recognized as a serious problem worldwide which affects both wild and cultured freshwater fish including the commercially important channel catfish (Ictalurus punctatus). The fundamental molecular mechanisms of the host immune response to the causative agent Flavobacterium columnare remain unclear, though gene expression analysis after the bacterial infection has been conducted. Alternative splicing, a post-transcriptional regulation process to modulate gene expression and increase the proteomic diversity, has not yet been studied in channel catfish following infection with F. columnare. In this study, genomic information and RNA-Seq datasets of channel catfish were used to characterize the changes of alternative splicing after the infection. Alternative splicing was shown to be induced by F. columnare infection, with 8.0% increase in alternative splicing event at early infection stage. Intriguingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced (DAS) gene sets after infection. This finding was consistent with our previous study in channel catfish following infection with Edwardsiella ictaluri. It was suggested to be a universal mechanism that genes involved in RNA binding and splicing were regulated to undergo differential alternative splicing after stresses in channel catfish. Moreover, many immune genes were observed to be differentially alternatively spliced after infection. Further studies need to be performed to get a deeper view of molecular regulation on alternative splicing after stresses, setting a foundation for developing catfish broodstocks with enhanced disease resistance.

GWAS Analysis Indicated Importance of NF-κB Signaling Pathway in Host Resistance Against Motile Aeromonas Septicemia Disease in Catfish.

Motile Aeromonas septicemia (MAS) disease caused by a bacterial pathogen, Aeromonas hydrophila, is an emerging but severe disease of catfish. Genetic enhancement of disease resistance is considered to be effective to control the disease. To provide an insight into the genomic basis of MAS disease resistance, in this study, we conducted a genome-wide association study (GWAS) to identify quantitative trait loci (QTL). A total of 1820 interspecific backcross catfish of 7 families were challenged with A. hydrophila, and 382 phenotypic extremes were selected for genotyping with the catfish 690 K SNP arrays. Three QTL on linkage group (LG) 2, 26 and 29 were identified to be significantly associated with MAS resistance. Within these regions, a total of 24 genes had known functions in immunity, 10 of which were involved in NF-κB signaling pathway, suggesting the importance of NF-κB signaling pathway in MAS resistance. In addition, three suggestively significant QTL were identified on LG 11, 17, and 20. The limited numbers of QTL involved in MAS resistance suggests that marker-assisted selection may be a viable approach for catfish breeding.

Identification of a novel C1q complement component in razor clam Sinonovacula constricta and its role in antibacterial activity.

The serum complement component C1q mediates a variety of immune regulatory functions. Herein, we identified a globular head C1q (ghC1q) gene in razor clam Sinonovacula constricta. The complete Sc-ghC1q gene was 872 bp long included an 81 bp 5'-untranslated region (UTR), a 95 bp 3'-UTR with a poly(A) tail, and an open reading frame (ORF) of 696 bp. The mRNA expression of Sc-ghC1q was upregulated in hepatopancreas and hemocytes. After Staphylococcus aureus or Vibrio anguillarum challenge, Sc-ghC1q mRNA transcript abundance was significantly upregulated in hemolymph. Recombinant Sc-ghC1q protein could bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and it could agglutinate both Gram-positive and Gram-negative bacteria. Additionally, flow cytometry revealed that Sc-ghC1q strongly promoted phagocytosis in hemocytes. Together, these results demonstrated that Sc-ghC1q played an important role in innate immunity in S. constricta.

Characterization of the ScghC1q-1 gene in Sinonovacula constricta and its role in innate immune responses.

C1q is an important immune gene that can mediate a variety of immune regulatory functions, and is involved in complement pathway activation. In the present study, a ghC1q gene from the razor clam Sinonovacula constricta was identified and named ScghC1q-1. The complete ScghC1q-1 gene is 692 bp in length, with an open reading frame (ORF) of 489 bp encoding a protein of 162 amino acids. ScghC1q-1 mRNA was widely expressed in various tissues, and transcript levels in the hemolymph were significantly up-regulated following Staphylococcus aureus or Vibrio anguillarum challenge. Recombinant ScghC1q-1 protein was found to agglutinate both Gram-positive and Gram-negative bacteria. These results indicate that ScghC1q-1 plays an essential role in the immune defense of S. constricta.

Heat stress induced alternative splicing in catfish as determined by transcriptome analysis.

Heat tolerance is increasingly becoming an important trait for aquaculture species with a changing climate. Transcriptional studies on responses to heat stress have been conducted in catfish, one of the most important economic aquaculture species around the world. The molecular mechanisms underlying heat tolerance is still poorly understood, especially at the post-transcriptional level including regulation of alternative splicing. In this study, existing RNA-Seq datasets were utilized to characterize the change of alternative splicing in catfish following heat treatment. Heat-tolerant and -intolerant catfish were differentiated by the time to lost equilibrium after heat stress. With heat stress, alternative splicing was generally increased. In heat-intolerant fish, the thermal stress induced 29.2% increases in alternative splicing events and 25.8% increases in alternatively spliced genes. A total of 282, 189, and 44 differential alternative splicing (DAS) events were identified in control-intolerant, control-tolerant, and intolerant-tolerant comparisons, corresponding to 252, 171, and 42 genes, respectively. Gene ontology analyses showed that genes involved in the molecular function of RNA binding were significantly enriched in DAS gene sets after heat stress in both heat-intolerant and -tolerant catfish compared with the control group. Similar results were also observed in the DAS genes between heat-intolerant and -tolerant catfish, and the biological process of RNA splicing was also enriched in this comparison, indicating the involvement of RNA splicing-related genes underlying heat tolerance. This is the first comprehensive study of alternative splicing in response to heat stress in fish species, providing insights into the molecular mechanisms of responses to the abiotic stress.