Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

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.

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.

Hemolytic reactions in the hemolymph of bivalve Sinonovacula constricta show complement-like activity.

The complement-like hemolysis method was used to determine the total complement-like activity of the plasma of Sinonovacula constricta. In this study, the effects of both physical and chemical conditions on complement hemolysis of S. constricta were measured. Physical conditions included proportion (S. constricta plasma: 2% rabbit red blood cells), temperature, time, and incubation, while the chemical factors consisted of Lipopolysaccharide (LPS), Flagellin (FLA), Zymosan, Peptidoglycan (PGN), Phenylmethanesulfonyl fluoride (PMSF), Methylamine, and Poly (I: C). The results showed that LPS, flagellin, Zymosan and PGN could activate complement-like activity of S. constricta plasma and cause hemolysis. PMSF and methylamine inhibited complement-like activity, resulting in the disappearance of hemolysis. Poly (I: C) had no effect on plasma complement-like activity. When the reaction temperature was less than 50 °C, hemolytic activity would increase following an increase in temperature. The ratio of plasma to rabbit blood cells had a great impact on the rate of hemolysis. Additionally, incubation with low speed oscillation could improve the hemolysis rate. It is indicated that the hemolytic reactions in the hemolymph of bivalve S. constricta show complement-like activity. The results contribute to further research on immune function of complement in bivalve.

Identification of a novel galectin in Sinonovacula constricta and its role in recognition of Gram-negative bacteria.

Galectins are soluble lectins that perform a pattern recognition function in invertebrate immunity and specifically recognise ß-galactoside residues via conserved carbohydrate recognition domains. However, their function in bivalve molluscs has received little attention. Herein, a galectin (ScGal2) in razor clam (Sinonovacula constricta) consisting of a 507 bp open reading frame encoding a protein of 168 amino acids was identified and characterised. The protein includes a carbohydrate recognition domain (CRD), and several residues involved in dimerisation were found. ScGal2 mRNAs were mainly detected in hemolymph and liver, and expression was upregulated significantly following challenge with Vibrio anguillarum. Recombinant rScGal2 protein displayed strong agglutination activity toward Gram-negative bacteria, and flow cytometry revealed that ScGal2 strongly promoted phagocytosis in hemocytes. These results suggest that ScGal2 plays an indispensable role in innate immunity in razor clam, and likely participates in immune recognition and clearance processes.

Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta.

Complement component 3 (C3) is central to the complement system, playing an important role in immune defense, immune regulation and immune pathology. Several C3 genes have been characterized in invertebrates but very few in shellfish. The C3 gene was identified from the razor clam Sinonovacula constricta, referred to here as Sc-C3. It was found to be highly homologous with the C3 gene of Ruditapes decussatus. All eight model motifs of the C3 gene were found to be included in the thiolester bond and the C345C region. Sc-C3 was widely expressed in all healthy tissues with expression being highest in hemolymph. A significant difference in expression was revealed at the umbo larvae development stage. The expression of Sc-C3 was highly regulated in the hemolymph and liver, with a distinct response pattern being noted after a challenge with Micrococcus lysodeikticus and Vibrio parahemolyticus. It is therefore suggested that a complicated and unique response pathway may be present in S. constricta. Further, serum of S. constricta containing Sc-C3 was extracted. This was activated by LPS or bacterium for verification for function. The more obvious immune function of Sc-C3 was described as an effective membrane rupture in hemocyte cells of rabbit, V. parahemolyticus and Vibrio anguillarum. Thus, Sc-C3 plays an essential role in the immune defense of S. constricta.

The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts.

Numerous studies have identified the detrimental effects for the biosphere of large plastic debris, the effect of microplastics (MPs) and nanoplastics (NPs) is less clear. The skin is the first point of contact with NPs, and skin fibroblasts have a vital role in maintaining skin structure and function. Here, a comparative approach is taken using three fibroblast cell lines from the zebrafish (SJD.1), human male newborn (BJ-5ta) and female adult (HDF/TERT164) and their response to polystyrene NP (PS-NPs) exposure is characterized. Cells were exposed to environmentally relevant PS-NP sizes (50, 500 and 1000 nm) and concentrations (0.001 to 10 µg/ml) and their uptake (1000 nm), and effect on cell viability, proliferation, migration, reactive oxygen species (ROS) production, apoptosis, alkaline phosphatase (ALP) and acid phosphatase (AP) determined. All fibroblasts took up PS-NPs, and a relationship between PS-NP particle size and concentration and the inhibition of proliferation and cell migration was identified. The inhibitory effect of PS-NPs on proliferation was more pronounced for human skin fibroblasts. The presence of PS-NPs negatively affected fibroblast migration in a time-, size- and concentration-dependent manner with larger PS-NPs at higher concentrations causing a more significant inhibition of cell migration, with human fibroblasts being the most affected. No major changes were detected in ROS production or apoptosis in NP challenged fibroblasts. While the ALP activity was increased in all fibroblast cell lines, only fish fibroblasts showed a significant increase in AP activity. The heterogeneous response of fibroblasts induced by PS-NPs was clearly revealed by the segregation of HDF, BJ.5ta and SJD.1 fibroblasts in principal component analysis. Our results demonstrate that PS-NP exposure adversely affected cellular processes in a cell-type and dose-specific manner in distinct fibroblast cell lines, emphasizing the need for further exploration of NP interactions with different cell types to better understand potential implications for human health.

Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca.

Family B1 G protein-coupled receptors (GPCRs) are one of the most well studied neuropeptide receptor families since they play a central role in many biological processes including endocrine, gastrointestinal, cardiovascular and reproduction in animals. The genes for these receptors emerged from a common ancestral gene in bilaterian genomes and evolved via gene/genome duplications and deletions in vertebrate and invertebrate genomes. Their existence and function have mostly been characterized in vertebrates and few studies exist in invertebrate species. Recently, an increased interest in molluscs, means a series of genomes have become available, and since they are less modified than insect and nematode genomes, they are ideal to explore the origin and evolution of neuropeptide gene families. This review provides an overview of Family B1 GPCRs and their peptide ligands and incorporates new data obtained from Mollusca genomes and taking a comparative approach challenges existing models on their origin and evolution.

Core genes of biomineralization and cis-regulatory long non-coding RNA regulate shell growth in bivalves.

INTRODUCTION: Bivalve molluscs are abundant in marine and freshwater systems and contribute essential ecosystem services. They are characterized by an exuberant diversity of biomineralized shells and typically have two symmetric valves (a.k.a shells), but oysters (Ostreidae), some clams (Anomiidae and Chamidae) and scallops (Pectinida) have two asymmetrical valves. Predicting and modelling the likely consequences of ocean acidification on bivalve survival, biodiversity and aquaculture makes understanding shell biomineralization and its regulation a priority. OBJECTIVES: This study aimed to a) exploit the atypical asymmetric shell growth of some bivalves and through comparative analysis of the genome and transcriptome pinpoint candidate biomineralization-related genes and regulatory long non-coding RNAs (LncRNAs) and b) demonstrate their roles in regulating shell biomineralization/growth. METHODS: Meta-analysis of genomes, de novo generated mantle transcriptomes or transcriptomes and proteomes from public databases for six asymmetric to symmetric bivalve species was used to identify biomineralization-related genes. Bioinformatics filtering uncovered genes and regulatory modules characteristic of bivalves with asymmetric shells and identified candidate biomineralization-related genes and lncRNAs with a biased expression in asymmetric valves. A shell regrowth model in oyster and gene silencing experiments, were used to characterize candidate gene function. RESULTS: Shell matrix genes with asymmetric expression in the mantle of the two valves were identified and unique cis-regulatory lncRNA modules characterized in Ostreidae. LncRNAs that regulate the expression of the tissue inhibitor of metalloproteinases gene family (TIMPDR) and of the shell matrix protein domain family (SMPDR) were identified. In vitro and in vivo silencing experiments revealed the candidate genes and lncRNA were associated with divergent shell growth rates and modified the microstructure of calcium carbonate (CaCO3) crystals. CONCLUSION: LncRNAs are putative regulatory factors of the bivalve biomineralization toolbox. In the Ostreidae family of bivalves biomineralization-related genes are cis-regulated by lncRNA and modify the planar growth rate and spatial orientation of crystals in the shell.

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.

Evolution and Potential Function in Molluscs of Neuropeptide and Receptor Homologues of the Insect Allatostatins.

The allatostatins (ASTs), AST-A, AST-B and AST-C, have mainly been investigated in insects. They are a large group of small pleotropic alloregulatory neuropeptides that are unrelated in sequence and activate receptors of the rhodopsin G-protein coupled receptor family (GPCRs). The characteristics and functions of the homologue systems in the molluscs (Buccalin, MIP and AST-C-like), the second most diverse group of protostomes after the arthropods, and of high interest for evolutionary studies due to their less rearranged genomes remains to be explored. In the present study their evolution is deciphered in molluscs and putative functions assigned in bivalves through meta-analysis of transcriptomes and experiments. Homologues of the three arthropod AST-type peptide precursors were identified in molluscs and produce a larger number of mature peptides than in insects. The number of putative receptors were also distinct across mollusc species due to lineage and species-specific duplications. Our evolutionary analysis of the receptors identified for the first time in a mollusc, the cephalopod, GALR-like genes, which challenges the accepted paradigm that AST-AR/buccalin-Rs are the orthologues of vertebrate GALRs in protostomes. Tissue transcriptomes revealed the peptides, and their putative receptors have a widespread distribution in bivalves and in the bivalve Mytilus galloprovincialis, elements of the three peptide-receptor systems are highly abundant in the mantle an innate immune barrier tissue. Exposure of M. galloprovincialis to lipopolysaccharide or a marine pathogenic bacterium, Vibrio harveyi, provoked significant modifications in the expression of genes of the peptide precursor and receptors of the AST-C-like system in the mantle suggesting involvement in the immune response. Overall, our study reveals that homologues of the arthropod AST-systems in molluscs are potentially more complex due to the greater number of putative mature peptides and receptor genes. In bivalves they have a broad and varying tissue distribution and abundance, and the elements of the AST-C-like family may have a putative function in the immune response.

The temptin gene of the clade Lophotrochozoa is involved in formation of the prismatic layer during biomineralization in molluscs.

The biomineralization mechanism of mollusc shell has been studied for a long time, but there is a lack of understanding about the relationship between the shell formation in vitro and the signaling system in vivo. In this study, we cloned a novel shell matrix protein gene (hc-temptin), which only be characterized as a water-borne protein pheromone of molluscs in previous studies, from the freshwater mussel Hyriopsis cumingii. By bioinformatics analysis we found that temptin was a gene unique to the clade Lophotrochozoa, and it exists in all mollusc taxa except Cephalopoda. The current data supported the premise that temptin was generated in the early emergence of molluscs and that it maintained a high mutation rate to evolve relative independently. The specificity of hc-temptin expression in the mantle tissue suggests its potential to participate in biomineralization. Its sequence contained typical Ca2+ binding sites. Our experiments involving the pearl formation process, damaged shell repair process, and RNAi experiment showed that hc-temptin was a shell matrix protein that plays an important role in formation of the prismatic layer. The results of this study provided new insights about the origin of the temptin gene and its role in molluscs.

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.

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.

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.

The calcitonin-like system is an ancient regulatory system of biomineralization.

Biomineralization is the process by which living organisms acquired the capacity to accumulate minerals in tissues. Shells are the biomineralized exoskeleton of marine molluscs produced by the mantle but factors that regulate mantle shell building are still enigmatic. This study sought to identify candidate regulatory factors of molluscan shell mineralization and targeted family B G-protein coupled receptors (GPCRs) and ligands that include calcium regulatory factors in vertebrates, such as calcitonin (CALC). In molluscs, CALC receptor (CALCR) number was variable and arose through lineage and species-specific duplications. The Mediterranean mussel (Mytilus galloprovincialis) mantle transcriptome expresses six CALCR-like and two CALC-precursors encoding four putative mature peptides. Mussel CALCR-like are activated in vitro by vertebrate CALC but only receptor CALCRIIc is activated by the mussel CALCIIa peptide (EC50 = 2.6 ×10-5 M). Ex-vivo incubations of mantle edge tissue and mantle cells with CALCIIa revealed they accumulated significantly more calcium than untreated tissue and cells. Mussel CALCIIa also significantly decreased mantle acid phosphatase activity, which is associated with shell remodelling. Our data indicate the CALC-like system as candidate regulatory factors of shell mineralization. The identification of the CALC system from molluscs to vertebrates suggests it is an ancient and conserved calcium regulatory system of mineralization.

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.

Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions.

Galectins are lectins possessing an evolutionarily conserved carbohydrate recognition domain (CRD) with affinity for ß-galactoside. The key role played by innate immunity in invertebrates has recently become apparent. Herein, a full-length galectin (ScGal) was identified in razor clam (Sinonovacula constricta). The 528 bp open reading frame encodes a polypeptide of 176 amino acids with a single CRD and no signal peptide. ScGal mRNA transcripts were mainly expressed in hemolymph and gill, and were significantly up-regulated following bacterial challenge. Recombinant rScGal protein binds to and aggregates various bacteria, and has affinity for peptidoglycan, lipoteichoic acid and d-galactose. The protein also stimulates hemocytes to phagocytose invading bacterial pathogens. ScGal is an important immune factor in innate immunity, and a small protein with multiple important functions.

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.

Expression of a novel complement C3 gene in the razor clam Sinonovacula constricta and its role in innate immune response and hemolysis.

Complement component 3 (C3) is a core component of the complement system, and directly participates in immune regulation and immune defense. Isoforms of C3 have been reported in several species of vertebrate, but invertebrates, and more specifically clams, have been less well studied. An isoform of C3, named ScC3-2, was identified in Sinonovacula constricta (Chinese razor clam). ScC3-2 included eight conserved regions, a thioester bond and two predicted junction sites (α-ß and α-γ). The gene was expressed in the liver, gill, foot, hemolymph, mantle, gonad and siphon tissues. The gene was significantly upregulated in umbo larvae, suggesting that initial larval immunity may develop in umbo larvae. Moreover, the ScC3-2 mRNA expression patterns after challenge with Vibrio parahemolyticus and Micrococcus lysodeikticus exhibited an obvious upregulation at 8 h in the hemolymph and at 4 h in the liver, respectively. Furthermore, ScC3-2 showed effective membrane rupture of heterologous rabbit erythrocytes. The ScC3-2 protein was located on the surface of the cells during the process of hemolysis. After a comparative analysis, we suggest that the major structure and function of ScC3 and ScC3-2 are analogous. Our findings suggest that ScC3-2 plays an important immune function, and an intricate complement response may exist in S. constricta.