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Self-nonself discrimination is a common theme for all of the organisms in different evolutionary branches, which is also the most fundamental step for host immune protection. Plenty of pattern recognition receptors (PRRs) with great diversity have been identified from different organisms to recognize various pathogen-associated molecular patterns (PAMPs) in the last two decades, depicting a complicated scene of host-pathogen interaction. However, the detailed mechanism of the complicate PAMPs–PRRs interactions at the contacting interface between pathogens and hosts is still not well understood. All of the cells are coated by glycosylation complex and thick carbohydrates layer. The different polysaccharides in extracellular matrix of pathogen-host are important for nonself recognition of most organisms. Coincidentally, massive expansion of PRRs, majority of which contain recognition domains of Ig, leucine-rich repeat (LRR), C-type lectin (CTL), C1q and scavenger receptor (SR), have been annotated and identified in invertebrates by screening the available genomic sequence. The phylum Mollusca is one of the largest groups in the animal kingdom with abundant biodiversity providing plenty of solutions about pathogen recognition and immune protection, which might offer a suitable model to figure out the common rules of immune recognition mechanism. The present review summarizes the diverse PRRs and common elements of various PAMPs, especially focusing on the structural and functional characteristics of canonical carbohydrate recognition proteins and some novel proteins functioning in molluscan immune defense system, with the objective to provide new ideas about the immune recognition mechanisms.

Biomineralization refers to the dynamic physiological processes whereby living organisms elaborate mineralized tissues. The existence of extremely abundant molluscan species implies the diversity of mineralized tissues, since the majority of them (Conchifera) produce shells that vary in size and shape. Over the past decades, great progress has been made on the study of the cellular biology of shell biomineralization. The construction of the molluscan shell is the archetype of a biologically controlled mineralization which requires specialized cellular machinery. It has been so far demonstrated that the cells involved in shell formation come from two different tissues: 1) outer mantle epithelial cells (OME) secrete the organic matrix, among which shell matrix proteins (SMPs) determine mineralogical and crystallographic properties of shell; and 2) circulating hemocytes which take part in the deposition of intracellular biominerals and deliver them to the mineralization sites. Mounts of novel SMPs have been identified by using molecular biology techniques (gene cloning, in situ hybridization, immunohistochemistry et al.) coupled with high-throughput sequencing data (genome, proteome, secretome and transcriptome) , and their corresponding functions during shell formation have also been confirmed. The cellular activity of OME and hemocytes during shell formation are significantly increased during shell regeneration process. A potential cellular basis model for molluscan shell formation is proposed. The shell matrix proteins, mostly secreted from OME, and a few secreted from hemocytes or other organs, are either directly delivered to the mineralization site via exosome or classical secretory pathway, or firstly transported to the hemolymph, and then engulfed by hemocytes (mainly granulocytes), which will disintegrate and release shell proteins and CaCO3 crystals at mineralization front. Besides, OME and hemocytes may be involved in the nucleation and remodeling process of CaCO3 mineralization. These cells and cell products work co-operatively to produce an organo-mineral shell, which is composed of various biomineral ultra-structures and macromolecular organic components.

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the “middle” stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), β-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.

DM9 domain containing protein (DM9CP) is a recently discovered novel pattern recognition receptor (PRR) with the ability to recognize various microbes, but its role as a cytosolic PRR to track the invading microbes and trigger signaling pathway is still not clear. In the present study, a DM9CP (designated as Cg DM9CP-6) and an intracellular regulatory molecule, 14-3-3 (designated as Cg 14-3-3ε) were identified from oyster Crassostrea gigas , which contained two tandem DM9 repeats and a 14-3-3ε domain, respectively. Cg DM9CP-6 and Cg 14-3-3ε were higher expressed in haemocytes, and their mRNA expression levels increased significantly after Vibrio splendidus stimulation. Cg DM9CP-6 could bind various polysaccharides (LPS, PGN, MAN, and D-mannose) and microbes ( Staphylococcus aureus , Micrococcus luteus , V. splendidus , Escherichia coli , Yarrowia lipolytica , and Pichia pastoris ) in vitro, and it was observed to be colocalized with the FITC-labeled V. splendidus , E. coli and Y. lipolytica in haemocytes in vivo. The pull-down, surface plasmon resonance (SPR) and Co-Immunoprecipitation (Co-IP) assays all demonstrated that Cg 14-3-3ε was able to interact with Cg DM9CP-6 in vitro or in vivo. After the expression of Cg DM9CP-6 and Cg 14-3-3ε was knocked down separately by RNAi, the nuclear translocation of Cg Rel in haemocytes was inhibited, and the mRNA expressions of interleukin17-3 ( Cg IL17-3), Cg IL17-6, Cg Lysozyme and Cg BigDef1 in haemocytes all decreased significantly after the oysters were stimulated with V. splendidus . The results collectively indicated that Cg DM9CP-6 could function as an intracellular PRR to be associated with Cg 14-3-3ε to trigger the NF-κB pathway, which eventually regulated the immune responses including the expressions of inflammatory cytokines and antimicrobial molecules in oysters.

Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as Cg GLS-1) was identified from Pacific oyster Crassostrea gigas , whose open reading frame was of 1836 bp. Cg GLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica . The enzyme activity of recombinant Cg GLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. Cg GLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. Cg GLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p  < 0.01) after lipopolysaccharide (LPS) stimulation. The Cg GLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of Cg GLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of Cg mGluR6, Cg AP-1, cytokines Cg IL17-5 and Cg TNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of Cg Caspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that Cg GLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines Cg IL17-5 and Cg TNF-1, as well as hemocyte apoptosis.

Background The deep-sea snail Phymorhynchus buccinoides belongs to the genus Phymorhynchus (Neogastropoda: Raphitomidae), and it is a dominant specie in the cold seep habitat. As the environment of the cold seep is characterized by darkness, hypoxia and high concentrations of toxic substances such as hydrogen sulfide (H 2 S), exploration of the diverse fauna living around cold seeps will help to uncover the adaptive mechanisms to this unique habitat. In the present study, a chromosome-level genome of P. buccinoides was constructed and a series of genomic and transcriptomic analyses were conducted to explore its molecular adaptation mechanisms to the cold seep environments. Results The assembled genome size of the P. buccinoides was approximately 2.1 Gb, which is larger than most of the reported snail genomes, possibly due to the high proportion of repetitive elements. About 92.0% of the assembled base pairs of contigs were anchored to 34 pseudo‐chromosomes with a scaffold N50 size of 60.0 Mb. Compared with relative specie in the shallow water, the glutamate regulative and related genes were expanded in P. buccinoides , which contributes to the acclimation to hypoxia and coldness. Besides, the relatively high mRNA expression levels of the olfactory/chemosensory genes in osphradium indicate that P. buccinoides might have evolved a highly developed and sensitive olfactory organ for its orientation and predation. Moreover, the genome and transcriptome analyses demonstrate that P. buccinoides has evolved a sulfite-tolerance mechanism by performing H 2 S detoxification. Many genes involved in H 2 S detoxification were highly expressed in ctenidium and hepatopancreas, suggesting that these tissues might be critical for H 2 S detoxification and sulfite tolerance. Conclusions In summary, our report of this chromosome-level deep-sea snail genome provides a comprehensive genomic basis for the understanding of the adaptation strategy of P. buccinoides to the extreme environment at the deep-sea cold seeps.

MicroRNAs (miRNAs) have emerged as key regulators in many pathological processes by suppressing the transcriptional and post-transcriptional expression of target genes. MiR-2008 was previously found to be significantly up-regulated in diseased sea cucumber Apostichopus japonicus by high-through sequencing, whereas the reads of miR-137, a well-documented tumor repressor, displayed no significant change. In the present study, we found that miR-137 expression was slightly attenuated and miR-2008 was significantly enhanced after Vibrio splendidus infection or Lipopolysaccharides application. Further target screening and dual-luciferase reporter assay revealed that the two important miRNAs shared a common target gene of betaine–homocysteine S-methyltransferase (AjBHMT), which exhibited noncorrelated messenger RNA and protein expression patterns after bacterial challenge. In order to fully understand their regulatory mechanisms, we conducted the functional experiments in vitro and in vivo. The overexpression of miR-137 in sea cucumber or primary coelomocytes significantly decreased, whereas the inhibition of miR-137 increased the mRNA and protein expression levels of AjBHMT. In contrast, miR-2008 overexpression and inhibition showed no effect on AjBHMT mRNA levels, but the concentration of AjBHMT protein displayed significant changes both in vitro and in vivo. Consistently, the homocysteine (Hcy) contents were also accordingly altered in the aberrant expression analysis of both miRNAs, consistent with the results of the AjBHMT silencing assay in vitro and in vivo. More importantly, small interfering RNA mediated AjBHMT knockdown and Hcy exposure analyses both significantly increased reactive oxygen species (ROS) production and decreased the number of surviving invasive pathogen in sea cucumber coelomocytes. Taken together, these findings confirmed the differential roles of sea cucumber miR-137 and miR-2008 in regulating the common target AjBHMT to promote ROS production and the clearance of pathogenic microorganisms through Hcy accumulation.

C-type lectins are a superfamily of Ca(2+) dependent carbohydrate-recognition proteins that play significant diverse roles in nonself-recognition and clearance of invaders. Though they are well characterized in vertebrates, the study of the potential function and mechanism of C-type lectins in invertebrate immunity is still in its infancy. A C-type lectin (CfLec-1) from scallop Chlamys farreri, a dominant cultured mollusk species in China, was selected to investigate its mRNA expression, localization and the possible functions in innate immunity in the present study. After scallop was stimulated by three typical PAMPs, the mRNA expression of CfLec-1 in hemocytes was poles apart. It was significantly up-regulated (p<0.01) after scallops were stimulated by LPS or β-glucan, but significantly down-regulated (p<0.01) after PGN stimulation. The binding ability of recombinant CfLec-1 (designated as rCfLec-1) towards eight PAMPs was investigated subsequently by PAMPs microarray, which revealed rCfLec-1 could bind LPS, PGN and mannan in vitro, indicating CfLec-1 served as a PRR involved in the pathogen recognition. Immunofluorescence assay with polyclonal antibody specific for CfLec-1 revealed that CfLec-1 was mainly located in the mantle and gill of the scallop. CfLec-1 could bind to the surface of scallop hemocytes and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-1 antibody. Meanwhile, rCfLec-1 could also enhance the phagocytic activity of scallop hemocytes against Escherichia coli. The results clearly suggested that CfLec-1 in C. farreri not only served as a PRR involved in the PAMPs recognition, but also functioned as an opsonin participating in the clearance of invaders. It is therefore suspected that CfLec-1 could be an attachment-molecule to nonself-agents acting as an alternative to immunoglobulin in vertebrates.

MicroRNAs (miRNAs) represent a class of small ncRNAs that repress gene expression on the post-transcriptional level by the degradation or translation inhibition of target mRNA. Three small RNA libraries from oyster haemocytes were sequenced on the Illumina platform to investigate the latent immunomodulation of miRNAs after bacteria challenge and heat stress. Totally, 10,498,663, 8,588,606 and 9,679,663 high-quality reads were obtained in the control, bacteria and bacteria+heat library, respectively, from which 199 oyster miRNAs including 71 known and 128 novel ones were identified. Among these miRNAs, 6 known and 23 novel ones were predicted to possess more than one precursor-coding region, and cgi-miR-10a, cgi-miR-184b, cgi-miR-100, cgi-miR-1984 and cgi-miR-67a were observed to be the most abundant miRNAs in the control library. The expression levels of 22 miRNAs in the bacteria library were significantly higher than those in the control library, while there were another 33 miRNAs whose expression levels were significantly lower than that in the control library. Meanwhile, the expression levels of 65 miRNAs in the bacteria+heat library changed significantly compared to those in the bacteria library. The target genes of these differentially expressed miRNAs were annotated, and they fell in immune and stress-related GO terms including antioxidant, cell killing, death, immune system process, and response to stimulus. Furthermore, there were 42 differentially expressed miRNAs detected in both control/bacteria and bacteria/bacteria+heat comparisons, among which 9 miRNAs displayed the identical pattern in the two comparisons, and the expression alterations of 8 miRNAs were confirmed using quantitative RT-PCR. These results indicated collectively that immune challenge could induce the expression of immune-related miRNAs, which might modulate the immune response such as redox reaction, phagocytosis and apoptosis, and the expression of some immune-related miRNAs could be also regulated by heat stress to improve the environmental adaption of oyster.

Neural cell adhesion molecule 1 (NCAM1/CD56) as a well-known surface marker for natural killer (NK) cells plays important roles in cell migration, adhesion, and inflammation. In the present study, NCAM1 homolog containingthree immunoglobulin domains, one fibronectin type 3 domain, a transmembrane region and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) was identified from the Pacific oyster, Crassostrea gigas (defined as Cg NCAM1). The mRNA transcripts of Cg NCAM1 were highly expressed in haemocytes. The mRNA expressions of Cg NCAM1 in haemocytes increased significantly after Vibrio splendidus stimulation. The positive green signals of Cg NCAM1 and SH2-containing inositol 5-phosphatase ( Cg SHIP2) could translocate onto the haemocyte membrane after V. splendidus stimulation. The recombinant extracellular domains of Cg NCAM1 exhibited binding activity towards various pathogen-associated molecular patterns (PAMPs) and microbes. Upon binding to its ligands, Cg NCAM1 recruited Cg SHIP2 to transduce inhibitor signals to reduce the phosphorylation of Cg P38. The inhibition of Cg P38 reduced the methylation of histone H3K4 and nuclear translocation of NF-κB, which eventually inhibited the mRNA expressions of inflammatory factors ( Cg IL17-2/3/6 and Cg TNF-2) to suppress inflammation. These results suggested that Cg NCAM1 could function as an immune checkpoint to sense different PAMPs and microbes and reduce the inflammation through inhibiting P38-epigenetic and P38-NF-κB pathways in oysters.