Wenguang Liu

Seawater Acidification and Elevated Temperature Affect Gene Expression Patterns of the Pearl Oyster Pinctada fucata

Oceanic uptake of anthropogenic carbon dioxide results in decrease in seawater pH and increase in temperature. In this study, we demonstrated the synergistic effects of elevated seawater temperature and declined seawater pH on gene expression patterns of aspein, calmodulin, nacrein, she-7-F10 and hsp70 in the pearl oyster Pinctada fucata. Under ‘business-as-usual’ scenarios, four treatments were examined: (1) ambient pH (8.10) and ambient temperature (27°C) (control condition), (2) ambient pH and elevated temperature (+3°C), (3) declined pH (7.70) and ambient temperature, (4) declined pH and elevated temperature. The results showed that under warming and acidic seawater conditions, expression of aspein and calmodulin showed no significant differences among different time point in condition 8.10 T. But the levels of aspein and calmodulin in conditions 8.10 T+3, 7.70 T and 7.70 T+3, and levels of nacrein, she-7-F10 in all the four treatments changed significantly. Low pH and pH×temperature interaction influenced the expression of aspein and calmodulin significantly after hours 48 and 96. Significant effects of low pH and pH×temperature interaction on the expression of nacrein were observed at hour 96. The expression level of she-7-F10 was affected significantly by pH after hours 48 and 96. The expression of hsp70 was significantly affected by temperature, pH, temperature×pH interaction at hour 6, and by temperature×pH interaction at hour 24. This study suggested that declined pH and pH×temperature interaction induced down regulation of calcification related genes, and the interaction between declined seawater pH and elevated temperature caused up regulation of hsp70 in P. facata. These results demonstrate that the declined seawater pH and elevated temperature will impact the physiological process, and potentially the adaptability of P. fucata to future warming and acidified ocean.

Molecular characterization of interferon regulatory factor 2 (IRF-2) homolog in pearl oyster Pinctada fucata.

Interferon regulatory factors (IRFs) control many facets of the innate and adaptive immune responses, regulate the development of the immune system itself and involve in reproduction and morphogenesis. In the present study, the IRF-2 homology gene, PfIRF-2 from pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfIRF-2 encodes a putative protein of 350 amino acids, and contains a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. Comparison and phylogenetic analysis revealed that PfIRF-2 shared a relatively higher identity with other mollusk but relatively lower identity with vertebrate IRF-2, and was clustered with IRF-1 subfamily composed of IRF-2 and IRF-1. Furthermore, gene expression analysis revealed that PfIRF-2 involved in the immune response to LPS and poly(I:C) stimulation. Immunofluorescence assay showed that the expressed PfIRF-2 was translocated into the nucleus and dual-luciferase reporter assays indicated that PfIRF-2 could involved and activate interferon signaling or NF-κB signal pathway in HEK293 cells. The study of PfIRF-2 may help better understand the innate immune in mollusk.

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata.

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

Seasonal variation of biochemical components in clam (Saxidomus purpuratus Sowerby 1852) in relation to its reproductive cycle and the environmental condition of Sanggou Bay, China

Seasonal variation of biochemical components in clam (Saxidomus purpuratus Sowerby 1852) was investigated from March 2012 to February 2013 in relation to environmental condition of Sanggou Bay and the reproductive cycle of clam. According to the histological analysis, the reproductive cycle of S. purpuratus includes two distinctive phases: a total spent and inactive stage from November to January, and a gametogenesis stage, including ripeness and spawning, during the rest of the year. Gametes were generated at a low temperature (2.1°C) in February. Spawning took place once a year from June to October. The massive spawning occurred in August when the highest water temperature and chlorophyll a level could be observed. The key biochemical components (glycogen, protein and lipid) in five tissues (gonad, foot, mantle, siphon and adductor muscle) were analyzed. The glycogen content was high before gametogenesis, and decreased significantly during the gonad development in the gonad, mantle and foot of both females and males, suggesting that glycogen was an important energy source for gonad development. The protein and lipid contents increased in the ovary during the gonad development, demonstrating that they are the major organic components of oocytes. The lipid and protein contents decreased in the testis, implying that they can provide energy and material for spermatogenesis. The results also showed that protein stored in the mantle and foot could support the reproduction after the glycogen was depleted.

Effect of ocean acidification on growth, calcification, and gene expression in the pearl oyster, Pinctada fucata

In this study, shell growth, shell microstructure, and expression levels of shell matrix protein genes (aspein, n16, and nacrein) that play a key role in the CaCO3 crystal polymorphism (calcite and aragonite) of the shell were investigated in the pearl oyster Pinctada fucata at pH 8.10, 7.70, and 7.40. We found that the shell length and total weight index did not vary significantly between oysters reared at pH 8.10 and 7.70, but was significantly lower at pH 7.40. Calcium content and shell hardness were not significantly different between pH 8.10 and 7.70, but were significantly different at pH 7.40. At pH 7.40, the shell exhibited a poorly organized nacreous microstructure, and showed an apparent loss of structural integrity in the nacreous layer. The prismatic layer appeared morphologically dissimilar from the samples at pH 8.10 and 7.70. The internal layer was corroded and had dissolved. At pH 7.40, the expression levels of nacrein, aspein, and n16 decreased on day 1, and remained low between days 2 and 42. The expression levels of these genes were significantly lower at pH 7.40 than at pH 8.10 and 7.70 during days 2-42. These results suggest that ocean acidification will have a limited impact on shell growth, calcification, and associated gene expression levels at a pH of 7.70, which is projected to be reached by the end of the century. The negative effects were found on calcification and gene expression occurred at the lowest experimental pH (7.40).

Effect of Temperature on Gene Expression in the Pearl Oyster Pinctada fucata

In this study, we examined the effect of elevated temperature on the expression patterns of genes, i.e., nacrein, irr, n16, n19, and hsp70 in the pearl oyster Pinctada fucata. The experiment was carried out at 4 temperatures, i.e., 20°C (ambient, control), 24, 28°C, and 32°C. The expression levels of target genes in P. fucata were assayed at 0, 6, 24, 48, and 96 h via real-time polymerase chain reaction. Results showed that the expression levels of nacrein and irr had no significant variations among different time points below 28°C, but significantly increased over time at 32°C. The expression levels of n16 and n19 did not change markedly at 20°C. The former increased significantly at 6 h and 24 h while the latter substantially decreased during 6–96 h at 24, 28 and 32°C. Among different temperatures, the level of n16 was significantly lower at 20°C than at other temperatures during 6–96 h, and the level of n19 significantly varied among different temperatures at 48 h and 96 h. The expression level of hsp70 was significantly higher at 32°C than at 20, 24 and 28°C at 24 h. These results demonstrated that elevated temperature impacted the physiological processes of P. fucata and potentially influenced its adaptability to thermal stress.

Proteome and Transcriptome Analysis of Ovary, Intersex Gonads, and Testis Reveals Potential Key Sex Reversal/Differentiation Genes and Mechanism in Scallop Chlamys nobilis

Bivalve mollusks exhibit hermaphroditism and sex reversal/differentiation. Studies generally focus on transcriptional profiling and specific genes related to sex determination and differentiation. Few studies on sex reversal/differentiation have been reported. A combination analysis of gonad proteomics and transcriptomics was conducted on Chlamys nobilis to provide a systematic understanding of sex reversal/differentiation in bivalves. We obtained 4258 unique peptides and 93,731 unigenes with good correlation between messenger RNA and protein levels. Candidate genes in sex reversal/differentiation were found: 15 genes differentially expressed between sexes were identified and 12 had obvious sexual functions. Three novel genes (foxl2, β-catenin, and sry) were expressed highly in intersex individuals and were likely involved in the control of gonadal sex in C. nobilis. High expression of foxl2 or β-catenin may inhibit sry and activate 5-HT receptor and vitellogenin to maintain female development. High expression of sry may inhibit foxl2 and β-catenin and activate dmrt2, fem-1, sfp2, sa6, Amy-1, APCP4, and PLK to maintain male function. High expression of sry, foxl2, and β-catenin in C. nobilis may be involved in promoting and maintaining sex reversal/differentiation. The downstream regulator may not be dimorphic expressed genes, but genes expressed in intersex individuals, males and females. Different expression patterns of sex-related genes and gonadal histological characteristics suggested that C. nobilis may change its sex from male to female. These findings suggest highly conserved sex reversal/differentiation with diverged regulatory pathways during C. nobilis evolution. This study provides valuable genetic resources for understanding sex reversal/differentiation (intersex) mechanisms and pathways underlying bivalve reproductive regulation.

Identification and analysis of an MKK4 homologue in response to the nucleus grafting operation and antigens in the pearl oyster, Pinctada fucata

ABSTRACT The mitogen‐activated protein kinase kinase 4 (MKK4) is a key component of the c‐Jun N‐terminal kinase (JNK) signaling pathway and regulates multiple cellular activities. However, little is known about the roles of this kinase in pearl oyster. In this study, we identified an MKK4 homologue in Pinctada fucata by using a transcriptome database. Sequence analysis and protein structure prediction showed that PfMKK4 is highly conserved to MKK4 from other vertebrate and invertebrate species. Phylogenetic analysis revealed that PfMKK4 has the closest relationship with that from Crassostrea gigas. QPCR was used to investigate expression profiles in different healthy adult tissues and developmental stages of P. fucata. We found that PfMKK4 was ubiquitously expressed in all tissues and developmental stages examined except for in D‐shaped larvae. Gene expression analysis suggested that PfMKK4 is involved in the response to the nucleus insertion operation. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [poly(I:C)] stimulation in vivo reduced PfMKK4 mRNA expression at 6h, 48h and 48h, 72h, respectively. LPS and poly(I:C) induced PfMKK4 phosphorylation in a primary mantle cell culture. These results contribute to better understanding of the potential role played by PfMKK4 in protecting the pearl oyster from injury caused by grafting or disease. HIGHLIGHTSPfMKK4 exhibits all the typical features of MKK4 gene.PfMKK4 wide distributes in tissues and developmental stages.PfMKK4 gene was expressed throughout the whole developmental stages, while the highest level was detected in the polar body stage.PfMKK4 may be involved in post‐graft processes.PfMKK4 is involved in immune response in Pinctada fucata.

Regulation of IL-17 by lncRNA of IRF-2 in the pearl oyster

&NA; Long noncoding RNAs (lncRNAs), once thought to be nonfunctional, have recently been shown to participate in the multilevel regulation of transcriptional, posttranscriptional and epigenetic modifications and to play important roles in various biological processes, including immune responses. However, the expression and roles of lncRNAs in invertebrates, especially nonmodel organisms, remain poorly understood. In this study, by comparing a transcriptome to the PfIRF‐2 genomic structure, we identified lncIRF‐2 in the PfIRF‐2 genomic intron. The results of the RNA interference (RNAi) and the nucleus grafting experiments indicated that PfIRF‐2 might have a negative regulatory effect on lncIRF‐2, and PfIRF‐2 and lncIRF‐2 may have a positive regulatory effect on PfIL‐17. Additionally, lncIRF‐2, PfIRF‐2 and PfIL‐17 were involved in responses to the nucleus graft. These results will enhance the knowledge of lncIRF‐2, IRF‐2, and IL‐17 functions in both pearl oysters and other invertebrates. HighlightsLncRNA of PfIRF‐2 gene (lncPfIRF‐2) was located in Pinctada fucata.LncIRF‐2 and PfIRF‐2 had effects on PfIL‐17.LncIRF‐2, PfIRF‐2 and PfIL‐17 involved in the nucleus grafting operation.

Molecular cloning and characterization of a putative mitogen-activated protein kinase (Erk1/2) gene: Involvement in mantle immunity of Pinctada fucata

&NA; Extracellular signal‐regulated kinases (ERKs) are conserved and related with protein‐serine/threonine kinases that participate in the regulation of multiple biological processes, such as cell survival, cell differentiation, proliferation, metabolism, and inflammation. However, little is known about the roles of this kinase in the pearl oyster. In this study, we cloned and identified an ERK homolog from Pinctada fucata (PfErk). Furthermore, we have unraveled its expressional kinetics after lipopolysaccharide (LPS) and polyinosinic‐epolycytidylic acid (poly I:C) immune challenge. Pferk harbored a 5′ untranslated region (UTR) of 12 bp, a coding sequence of 1074 bp, and a 3′ UTR of 882 bp. The putative peptide comprised a predicted molecular mass of 41.19 kDa, with a theoretical pI of 6.15. Sequence analysis showed that it possesses one STK catalytic domain and a conserved His‐Arg‐Asp (HRD) domain. In addition, a canonical Thr‐Glu‐Tyr (TEY) dual phosphorylation motif and an ATRW substrate binding site were also identified in the coding protein. Homology assessment of PfErk showed high similarity to Homo sapiens ERK. Phylogenetic analysis supported a close evolutionary relationship with molluscan orthologs. The expression patterns of Pferk were observed in seven different tissues of pearl oyster, with highest expression in the mantle and lowest expression in the digestive gland. Pferk mRNA expression levels were detected at developmental stages, with the highest expression in D‐shaped larvae, followed by the 32‐cell stage. The mRNA expression of Pferk was upregulated significantly in P. fucata mantle primary cells and mantle tissue after LPS and poly (I:C) treatment, and PfErk phosphorylation levels were activated by LPS and poly (I:C) challenges. Overall, our results suggested that PfErk may play important roles in pearl oyster innate immunity, and provided a new understanding of mantle immunity in the pearl oyster. HighlightsA novel Erk was first identified from P. fucata.PfErk shared a significant homology with other mollusk ERK proteins.PfErk wide was expressed in all examined tissues and developmental stages.PfErk was involved in mantle immunity in vivo and in vitro.