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.info:eu-repo/semantics/publishedVersio

Effects of Alkalinity and pH on Survival, Growth, and Enzyme Activities in Juveniles of the Razor Clam, Sinonovacula constricta

In order to clarify the possibility of rearing razor clams (Sinonovacula constricta) in inland saline water (ISW) and to facilitate their breeding under these stressful conditions, we performed semi-static acute and chronic toxicity tests to determine the effects of carbonate alkalinity (CA) and pH on the survival and growth rate, and critical metabolic enzyme activity in juvenile of S. constricta (JSC). (1) Acute toxicity test. As the water CA increased from 1.22 to 45.00 mmol L-1, the survival rate decreased significantly, which was exacerbated by the increase in the pH. When the water CA was set at 2.5 mmol L-1, the 48 h lethal concentration 50% (LC50) for JSCs with respect to pH was 9.86. When the water pH was 9.0, 9.5, and 10.0, the 48 h LC50 values for JSCs with respect to CA were 10.38, 8.79, and 3.11 mmol L-1, respectively. (2) Chronic toxicity test. Four experimental groups comprising the control, CAS, pHS, and CA-pHS were designated according to the target ISW data. After 3 months of stress, the JSC survival rate in each group exceeded 85%, but survival was significantly lower in the CA-pHS group than the control group (p < 0.05) in the first month. For the JSCs in various groups, the shell length growth rate (SGR) and weight gain (WG) rate were significantly lower in the CA-pHS group than the other groups (p < 0.05 for SGR; p < 0.001 for WG) in the first month. However, the difference in the growth rate among groups decreased in the next 2 months. For the JSCs in the CA-pHS group, the oxygen consumption, ammonia-N excretion, Na+/K+-ATPase, aspartate aminotransferase, and superoxide dismutase levels were significantly higher than those in the other groups during the first month, but there were no significant differences between the groups subsequently. The acetylcholinesterase and lysozyme levels did not differ significantly among groups during stress for 3 months. The integrated biomarker response index showed that stressors comprising high pH and CA could be tolerated well by JSCs over long periods of stress. These results indicate that water CA and pH together affect the survival, growth, and physiological activity of JSCs. S. constricta is suitable for culture in ISW

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.</i

Polymorphisms in the FOXO gene are associated with growth traits in the Sanmen breeding population of the razor clam Sinonovacula constricta

We identified the FOXO gene and five single-nucleotide polymorphisms (SNPs) in its coding region in the Sanmen breeding population of the razor clam Sinonovacula constricta. The ScFOXO gene encodes a protein of 625 amino acids in length that contains a conserved DNA-binding domain. Real-time polymerase chain reaction analysis showed that ScFOXO mRNA is widely expressed in adult tissues, with higher expression in the siphons and gills. Of the SNPs, c.879G > C (Val293Val) and c.1725A > G (Ser575Ser) are synonymous mutations, and c.543C > T (Phe181Leu), c.848A > G (Tyr283Cys), and c.1625G > C (Gly542Phe) are non-synonymous mutations. The SNPs all show significant associations with total body weight, shell length, shell width, and shell height (SH) in this population. Polymorphic parameter analysis showed that all SNPs except c.848A > G (Tyr283Cys) exhibit complete linkage. The SNP c.1625G > C (Gly542Phe) was used for genetic linkage mapping and demonstrates the strongest linkage (logarithm of the odds [LOD] value: 41.46) with the marker 96616 (37.02 cM) in the linkage group 9. The LOD value of this marker in relation to growth traits ranges from 1.04 to 1.53; only markers with a LOD value ≥ 3 were considered to be linked with growth traits in the razor clam linkage map. In summary, ScFOXO may be a growth trait-related gene and may represent a marker for Sanmen breeding group selection. Keywords: Sinonovacula constricta, Single-nucleotide polymorphism (SNP), FOXO, Growth trait

Characterization of two splice variants of EGFR and their effects on the growth of the razor clam

The epidermal growth factor receptor (EGFR) plays a vital role in cell growth, proliferation, and body growth. In this study, two EGFR isoforms (Sc-EGFR-1a and Sc-EGFR-1b) were obtained from Sinonovacula constricta. Sequence analysis of the coding region of Sc-EGFRs, revealed the 2 isoforms were generated from a common gene by alternative splicing and that one form possessed an extra 81 bp that corresponded to an insertion of 27 amino acids in the C-terminus. Expression of the Sc-EGFR isoforms were detectable in the early embryo stages and increased in the middle-late stages. Both isoforms were widely expressed in tissues, but the highest levels were detected in the siphon. Although both isoforms had a typical extracellular region, transmembrane region, and intracellular region, the insertion/deletion of 27 aa in the C-terminus changed the phosphorylation sites and may have influenced downstream kinase activities. In vitro, the proliferating cell nuclear antigen indicator and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that HEK293T cells transfected with Sc-EGFR-1a and Sc-EGFR-1b exhibited different proliferation patterns. Sc-EGFR-1a caused more rapid proliferation than Sc-EGFR-1b and the EGFP-N1 control. In vivo, Sc-EGFRs were successfully knocked-down for at least 15 days by injecting the clams with the dsRNA and was associated with a significant reduction in shell length. Our results reveal for the first time in mollusks the existence of alternative splicing of EGFR and provide the basis for further studies to establish the role of EGFR in growth and development of S. constricta. Keywords: Sinonovacula constricta, EGFR, Alternative splicing, Cell proliferatio

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

Dopamine beta-hydroxylase (D beta H) plays an essential role in the synthesis of catecholamines (CA) in neuroen-docrine networks. In the razor clam, Sinonovacula constricta a novel gene for D beta H (ScD beta H-alpha) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScD beta H-alpha gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScD beta H-alpha 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 ScD beta H-alpha 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 ScD beta H-alpha knock-down and transforming growth factor beta (TGF-beta 1) (P < 0.001) 48 h and 72 h after ScD beta H-alpha knock-down. Taken together the results suggest that the novel ScD beta H-alpha gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.National Key R & D Plan "Blue Granary Science and Technology Innovation" special project [2019YFD0900400]; Jiangsu Key Laboratory of Marine Biotechnology [HS2019002]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31472278

Expression and characterization of insulin-like growth factor II mRNA binding protein in the razor clam Sinonovacula constricta

Insulin-like growth factor-II mRNA binding protein (igf2bp) is an mRNA binding protein that regulates the post-transcriptional fate of multiple transcripts. In this study, a novel igf2bp gene was identified in the razor clam Sinonovacula constricta. The complete 2254 bp Sc-igf2bp cDNA consists of an open reading frame (ORF) encoding 610 amino acid (aa) residues. The deduced protein includes a single RNA recognition motif (RRM) and four K homology (KH) domains. Sc-igf2bp transcripts were expressed widely during different developmental stages and in adult tissues, and a 95 kDa recombinant pEGX-4T-1-Sc-igf2bp protein was expressed in Escherichia coli Rosetta (DE3) pLysS cells and purified using Glutathione Sepharose 4B resin. Activity was evaluated by GST pull-down assays, and mass spectrometry results indicated that Sc-igf2bp may participate in RNA-related processes. The findings provide a basis for further research on the role of igf2bp in regulating the growth and development of the razor clam

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

Dopamine beta-hydroxylase (D beta H) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The D beta H gene was identified from the razor clam Sinonovacula constricta and referred to as ScD beta H. The ScD beta H gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScD beta H transcript expression was abundant in liver and hemolymph. During early development, ScD beta H expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of D beta H were screened. After challenge with D beta H 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, ScD beta H may play an important regulating role in larval metamorphosis and juvenile growth.National Key R&D Program of China [2019YFD0900400]National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31472278]info:eu-repo/semantics/publishedVersio