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.CCMAR UIDB/04326/2020; CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER-022121. FCT: DL57/2016/CP1361/CT0020info:eu-repo/semantics/publishedVersio

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

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

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

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

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 &lt; 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 &lt; 0.05 for SGR; p &lt; 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

Evolução e função potencial em moluscos de neuropeptídeos e receptor homologues das allatostatinas de insetos

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.info:eu-repo/semantics/publishedVersio