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  • Dopamine beta-hydroxylase and its role in regulating the growth and larval metamorphosis in Sinonovacula constricta
    Publication . Li, Zhi; Niu, Donghong; Peng, Maoxiao; Xiong, Ya; Ji, Jie; Dong, Zhiguo; Li, Jiale
    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.
  • The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts
    Publication . Peng, Maoxiao; Félix, Rute C.; Canario, Adelino; Power, Deborah
    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 mu g/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 PSNPs, 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 celltype 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.
  • Domain-dependent evolution explains functional homology of protostome and deuterostome complement C3-like proteins
    Publication . Peng, Maoxiao; Li, Zhi; Cardoso, João; Niu, Donghong; Liu, Xiaojun; Dong, Zhiguo; Li, Jiale; Power, Deborah
    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.
  • Inland alkaline brackish water aquaculture of juvenile razor clam: survival, growth, physiology and immune responses
    Publication . Peng, Maoxiao; Li, Zhi; Liu, Xiaojun; Niu, Donghong; Li, Jiale
    In this study, we investigated rearing and breeding razor clam (Sinonovacula constricta) in inland alkaline brackish water. During 45 days of the experiment in candidate target inland area brackish water (TBW), survival, growth, enzyme activity and immune responses were analysed. The results showed that shell length and weight were significantly inhibited in the TBW group (p <0.001). Although the cumulative mortality was 81.479 ± 7.028 (p < 0.001) at 15 days, it increased slowly to 92.915 ± 2.271 (p < 0.001) at 45 days. The enzyme activities of Na+/K+-ATPase, aspartate aminotransferase and superoxide dismutase peaked at 15 days, and juvenile S. constricta (JSC) may engaged in partial anaerobic metabolism or damage to gill tissue, which may explain the high mortality rate at this time. Additionally, the phagocytic ability of haemocytes was inhibited (p < 0.001), but the metabolic activity was enhanced (p < 0.001). This implies that 15 days was the peak of TBW stress, and stress gradually decreased by days 30 and 45. TBW affected metabolism, osmotic regulation, and immune responses. There was an independent ionic interaction perhaps effect on JSC, primarily through Na+/K+ rate. Approximately 7 % of animals adapted successfully to TBW after 45 days. In summary, S. constricta has a great potential in further anti-TBW conditions selective breeding research.
  • The calcitonin-like system is an ancient regulatory system of biomineralization
    Publication . Cardoso, João CR; Félix, Rute C.; Ferreira, Vinícius; Peng, Maoxiao; Zhang, Xushuai; Power, Deborah
    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.
  • RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams
    Publication . Li, Zhi; Peng, Maoxiao; Power, Deborah; Niu, Donghong; Dong, Zhiguo; Li, Jiale
    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.
  • Evolution and potential function in molluscs of neuropeptide and receptor homologues of the insect allatostatins
    Publication . Li, Zhi; Cardoso, João; Peng, Maoxiao; Inácio, João P. S.; Power, Deborah
    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.
  • Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii
    Publication . Sousa, Carmen; Peng, Maoxiao; Guerreiro, Pedro; Cardoso, João; Chen, Liangbiao; Canario, Adelino; Power, Deborah
    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 seleniumrelated 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.
  • Revisiting the evolution of family B1 GPCRs and ligands: insights from mollusca
    Publication . Cardoso, João; Mc Shane, Jennifer; Li, Zhi; Peng, Maoxiao; Power, Deborah Mary
    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.
  • Evolution of chitin-synthase in molluscs and their response to ocean acidification
    Publication . Cardoso, João; Power, Deborah Mary; Peng, Maoxiao
    Chitin-synthase (CHS) is found in most eukaryotes and has a complex evolutionary history. Research into CHS has mainly been in the context of biomineralization of mollusc shells an area of high interest due to the consequences of ocean acidification. Exploration of CHS at the genomic level in molluscs, the evolution of isoforms, their tissue distribution, and response to environmental challenges are largely unknown. Exploiting the extensive molecular resources for mollusc species it is revealed that bivalves possess the largest number of CHS genes (12-22) reported to date in eukaryotes. The evolutionary tree constructed at the class level of molluscs indicates four CHS Type II isoforms (A-D) probably existed in the most recent common ancestor, and Type II-A (Type II-A1/Type II-A-2) and Type II-C (Type II-C-1/Type II-C-2) underwent further differentiation. Non-specific loss of CHS isoforms occurred at the class level, and in some Type II (B-D groups) isoforms the myosin head domain, which is associated with shell formation, was not preserved and highly species-specific tissue expression of CHS isoforms occurred. These observations strongly support the idea of CHS functional diversification with shell biomineralization being one of several important functions. Analysis of transcriptome data uncovered the species-specific potential of CHS isoforms in shell formation and a species-specific response to ocean acidification (OA). The impact of OA was not CHS isoform-dependent although in Mytilus, Type I-B and Type II-D gene expression was down-regulated in both M. galloprovincialis and M. coruscus. In summary, during CHS evolution the gene family expanded in bivalves generating a large diversity of isoforms with different structures and with a ubiquitous tissue distribution suggesting that chitin is involved in many biological functions. These findings provide insight into CHS evolution in molluscs and lay the foundation for research into their function and response to environmental changes.