Haitao Ma

A novel p38 MAPK indentified from Crassostrea hongkongensis and its involvement in host response to immune challenges

p38 mitogen-activated protein kinases (MAPKs) are conserved serine/threonine-specific kinases that are activated by various extracellular stimuli and play crucial regulatory roles in immunity, development and homeostasis. However, the function of p38s in mollusks, the second most diverse group of animals, is still poorly understood. In this study, a novel molluscan p38 (designated Chp38) was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA encoded a putative protein of 353 amino acids with a calculated molecular weight of approximately 40.3kDa. Similar to other reported p38 family proteins, the deduced Chp38 sequence contained a conserved dual phosphorylation TGY motif and a substrate binding site of ATRW. Phylogenetic analysis revealed that Chp38 was closest to its homolog from the Pacific oyster and belonged to the mollusk cluster. Quantitative real-time PCR analysis showed that Chp38 was constitutively expressed in all examined oyster tissues and developmental stages and that its expression in hemocytes was significantly up-regulated after pathogen (Vibrio alginolyticus and Staphylococcus haemolyticus) and PAMP (lipopolysaccharide and peptidoglycan) infections. Moreover, overexpression analysis revealed that Chp38 was localized in both the cytoplasm and nucleus of HEK293T cells and that it could significantly enhance AP-1 reporter gene activation in a dose-dependent manner. Altogether, these results provide the first experimental evidence of a functional p38 in oysters and suggest its involvement in the innate immunity of C. hongkongensis.

Development of 20 novel microsatellite markers in the Hong Kong oyster, Crassostrea hongkongensis

Twenty microsatellite markers were developed for the Hong Kong oyster, Crassostrea hongkongensis, and their polymorphisms were examined in a population. Allele numbers varied from 4 to 19 with an average of 9.35 per locus; observed heterozygosities fluctuated from 0.125 to 0.833 averaging 0.520, and the expected values ranged from 0.233 to 0.938 with an average of 0.664. Fifteen loci accorded statistically with Hardy–Weinberg equilibrium, while others showed a significant departure from the equilibrium, apparently due to heterozygote deficiency. Additionally, two pairs of loci showed significant linkage disequilibrium. Possible causes for null alleles were also discussed. In despite of null alleles, this set of microsatellite loci would be useful for genetic studies in C. hongkongensis.

Isolation and characterization of twenty-three microsatellite loci in the noble scallop, Chlamys nobilis

Twenty-three polymorphic microsatellite loci were developed from the noble scallop Chlamys nobilis, with an enriched partial genomic library. Polymorphisms of these loci were evaluated in a wild population of 30 individuals. The allele number of these 23 markers ranges from 2 to 14 with an average of 6.304 per locus. The observed and expected heterozygosity values range from 0.033 to 0.833 (averaging 0.494) and 0.033 to 0.895 (averaging 0.666), respectively. Fourteen loci accord with Hardy–Weinberg equilibrium, and the rest nine significantly deviate from HWE after Bonferroni correction. Additionally, three pairs of loci show significant linkage disequilibrium. These polymorphic markers would be useful in the future for studies of population genetics, linkage mapping and other relevant research in C. nobilis.

Cloning, characterization and comparative analysis of four death receptorTNFRs from the oyster Crassostrea hongkongensis

Apoptosis plays an important role in homeostasis of the immune systems. The tumor necrosis factor receptors (TNFRs) play critical roles in the extrinsic apoptosis pathways and in determining cell fate. In this study, four death receptors (DR) named ChEDAR, ChTNFR27, ChTNFR5, and ChTNFR16 were identified from the oyster Crassostrea hongkongensis. These ChDRs proteins had 382, 396, 414 and 384 amino acids, respectively, with the typical domains of death receptors, such as the signal peptide (SP), transmembrane helix region (TM) and death domains. Phylogenetic analysis showed that the ChDR proteins clustered into three distinct groups, indicating that these subfamilies had common ancestors. mRNA expression of the ChDRs were detected in all 8 of the selected oyster tissues and at different stages of development. Furthermore, expression of all the genes was increased in the hemocytes of oysters challenged with pathogens or air stress. Fluorescence microscopy revealed that the full-length proteins of the ChDRs were located in the plasma membrane of HEK293T cells. Over-expression of the ChDRs activated the NF-κB-Luc reporter in HEK293T cells in a dose-dependent manner. These results indicate that the ChDRs may play important roles in the extrinsic apoptotic pathways in oysters.

A molluscan extracellular signal-regulated kinase is involved in host response to immune challenges in vivo and in vitro

ABSTRACT Extracellular signal‐regulated kinases (ERKs) are a group of highly conserved serine/threonine‐specific protein kinases that function as important signaling intermediates in mitogen‐activated protein kinase (MAPK) pathways, which are involved in a wide variety of cellular activities, including proliferation, inflammation and cytokine production. However, little is known about the roles of this kinase in mollusk immunity. In this study, we identified a molluscan ERK homolog (ChERK) in the Hong Kong oyster (Crassostrea hongkongensis) and investigated its biological functions. The open reading frame (ORF) of ChERK encoded a polypeptide of 365 amino acids, with a predicted molecular weight of 41.96 kDa and pI of 6.43. The predicted ChERK protein contained typical characteristic motifs of the ERK family, including a dual threonine‐glutamate‐tyrosine (TEY) phosphorylation motif and an ATRW substrate binding site. Phylogenetic analysis revealed that ChERK belonged to the mollusk cluster and shared a close evolutionary relationship with ERK from Crassostrea gigas. In addition, quantitative real‐time PCR analysis revealed that ChERK expression was detected in all of the examined tissues and stages of embryonic development; its transcript level was significantly induced upon challenge with bacterial pathogens (Vibrio alginolyticus and Staphylococcus haemolyticus) in vivo and PAMPs (lipopolysaccharide and peptidoglycan) in vitro. Moreover, ChERK was mainly located in the cytoplasm of HEK293T cells. Taken together, these findings may provide novel insights into the functions of molluscan ERKs, especially their roles in response to immune challenge in oyster. HighlightsA novel oyster ERK was first identified from Crassostrea hongkongensis.ChERK shared a significant homology with other mollusk ERK proteins.ChERK was constitutively expressed in all examined oyster tissues and developmental stages.ChERK expression was significantly induced in response to bacterial pathogens and PAMPs challenges.ChERK was found to be located mainly in the cytoplasm of HEK293T cells.

The first invertebrate NFIL3 transcription factor with role in immune defense identified from the Hong Kong oyster, Crassostrea hongkongensis

Abstract NFIL3 (nuclear factor interleukin 3‐regulated) is a basic leucine zipper type transcription factor that mediates a variety of immune responses in vertebrates. However, the sequence information and function of NFIL3 homologs in invertebrates, especially mollusks, remains unknown. In the present study, the first NFIL3 homolog was identified in a marine mollusk, Crassostrea hongkongensis (designated as ChNFIL3), followed by its functional characterization. The full‐length cDNA of ChNFIL3 is 2221 bp and consists of an open reading frame (ORF) of 1536 bp that encodes a polypeptide of 551 amino acids. Simple Modular Architecture Research Tool (SMART) analysis indicated that ChNFIL3 has two basic leucin zipper domains, similar to the other known NFIL3 family proteins. Tissue distribution analysis of NFIL3 in this mollusk revealed high expression in digestive glands and hemocytes. A significant induction in the mRNA level of ChNFIL3 was observed following bacterial stimulation. ChNFIL3 was found to be localized in the nucleus and over expression of ChNIFL3 led to upregulation of transcriptional activity of an NF‐&kgr;B reporter gene in HEK 293T cells, indicating its role in innate immunity. Furthermore, addition of exogenous recombinant ChNFIL3 proteins resulted in enhanced mRNA level of hemocyte interleukin 17 in vitro. In conclusion, our findings revealed that NFIL3 in molluscs, plays a conserved role in host defense, similar to its mammalian homolog. HighlightsChNFIL3 was involved in the bacterial infection of oysters.ChNFIL3 could enhance the expression of interleukin 17 in vitro.Over expression of ChNFIL3 resulted in up‐regulated the transcriptional activity of an NF‐&kgr;B reporter gene.

The complete mitochondrial genome sequence of the giant clam Tridacna derasa (Tridacnidae: Tridacna)

Abstract The complete mitochondrial genome of the giant clam Tridacna derasa was completely sequenced by high-throughput sequencing method. The total length of the complete mitogenome was 20,760bp, including 13 protein-coding genes, 23 transfer RNA genes, 2 ribosomal RNA genes and a non-coding control region. The base composition of the genome is 28.41% A, 36.92% G, 21.94% G and 12.74% C with a total GC content of 34.68%. Phylogenetic tree based on complete mitogenome sequences revealed that T.derasa was closely related to T.squamosa, both belonging to the Tridacna genus.

A Lysin motif (LysM)-containing protein from Hong Kong oyster, Crassostrea hongkongensis functions as a pattern recognition protein and an antibacterial agent

Lysin motif (LysM)-containing proteins are a family of carbohydrate-binding modules and are generally regarded as chitin- and peptidoglycan-binding proteins. In the present study, a novel LysM-containing protein, designated as ChLysM, was cloned and identified in a marine mollusk, Crassostrea hongkongensis. The full-length cDNA of ChLysM consists of 1129 bp, with an open reading frame of 861 bp encoding a 286 amino acid polypeptide. The deduced protein had a calculated molecular mass of 32.66 kDa and a pI of 8.16. SMART analysis indicated that ChLysM has one Lysin motif and a transmembrane region in the C-terminal residues. Tissue distribution analysis of ChLysM revealed high expression in gills and hemocytes. The upregulated transcripts of ChLysM in response to bacterial challenge suggest that ChLysM is involved in innate immunity against pathogen infection. The recombinant protein of ChLysM was found to bind to various kinds of peptidoglycans from Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae, as well as binding strongly to both Gram-positive and Gram-negative bacteria. Moreover, ChLysM displayed broad-spectrum antibacterial activity against both Gram-positive bacteria (S. aureus and S. haemolyticus) and Gram-negative bacteria (Escherichia coli and Vibrio alginolyticus). Collectively, these results indicate that ChLysM is a pattern recognition molecule with bacterial growth-inhibiting activity in immune defense of C. hongkongensis.

The Molecular Mechanism Underlying Pro-apoptotic Role of Hemocytes Specific Transcriptional Factor Lhx9 in Crassostrea hongkongensis

Hemocytes are the central organ of immune defense against pathogens by means of inflammation, phagocytosis, and encapsulation in mollusks. The well-functioning of the host immune system relies on the hemocytes’ task exertion and frequent renewal, but the underlying renewal mechanism remains elusive at the gene level. Here, we identified one transcription factor, LIM homeobox 9, in Crassostrea hongkongensis (ChLhx9) that could be involved in hemocyte apoptosis or renewal. ChLhx9 contains a homeodomain and two LIM domains. The expression profile of ChLhx9 showed that it was specific and had high expression in hemocytes, and it significantly increased under the bacterial challenge. RNA interference of ChLhx9 dramatically decreased the apoptosis rate of hemocytes when compared with a control group, which strongly implies its pro-apoptotic role in hemocytes. Furthermore, the genomic responses to the knockdown of ChLhx9 were examined through RNA-seq, which showed that multiple pathways associated with cell apoptosis, including the apoptosis pathway, hippo signal pathway and p53 signaling pathway, were significantly down-regulated. Meanwhile, seven of the key apoptotic genes were confirmed to be upregulated by ChLhx9, among which ChASPP1 (apoptosis stimulating protein of p53) was confirmed to induce hemocyte apoptosis strongly, which demonstrates that ChASPP1 was a downstream target mediated by ChLhx9 that caused apoptosis. In conclusion, tissue-specific transcription factor ChLhx9 induces hemocyte apoptosis through activating apoptotic genes or pathways, which could contribute to hemocyte renewal and immune defense in oysters.

Analysis of in situ Transcriptomes Reveals Divergent Adaptive Response to Hyper- and Hypo-Salinity in the Hong Kong Oyster, Crassostrea hongkongensis

Crassostrea hongkongensis, a commercially valuable aquaculture species dwelling in estuaries along the coast of the South China Sea, is remarkable for its eurysalinity traits that enable its successful colonization of diverse osmotic niches ranging from near freshwater to seawater. In order to elucidate how this oyster copes with coastal waters with immense salinity differences, we performed in situ transcriptomic analysis (RNA-seq) to characterize the global expression patterns of oysters distributed across naturally formed salinity gradients in Zhenhai Bay along the northern coast of the South China Sea. Principal component analysis reveals distinct expression profiles of oysters living in the extreme conditions of hypo-salinity and hyper-salinity. Compared with the situation of optimal salinity for oyster growth, hypo-salinity mainly regulated expression of genes involved in FoxO and oxytocin signaling, tight junction and several immune pathways, while hyper-salinity altered gene expression implicated in amino acid metabolism, AMPK and PI3K-AKt signaling pathways, demonstrating the complexity and plasticity of transcriptomic expression underpinning oyster eurysalinity. Furthermore, the expression patterns of several genes correlated with salinity gradients reveals the fine-tuned coordination of molecular networks necessary for adaptive homeostasis in C. hongkongensis. In conclusion, a striking capacity and distinct patterns of transcriptomic expression contribute to eurysalinity adaptation in C. hongkongensis, which provides new mechanistic insights into the adaptive plasticity and resilience of marine mollusks.