Molecular identification of a novel antimicrobial peptide in giant Triton snail Charonia tritonis: mRNA profiles for tissues and its potential antibacterial activity.

Antimicrobial peptides (AMPs) play an important role in innate immunity against microorganisms. AMPs is an effective antibacterial agent, and the chances of causing pathogens to develop is very low. However, there is little information about AMPs in the giant Triton snail Charonia tritonis. In this research, an antimicrobial peptide gene (termed Ct-20534) was identified in C. tritonis. The open reading frame of Ct-20534 is 381 bp in size and it encodes a basic peptide precursor containing 126 amino acids. Ct-20534 gene was found to be expressed in all five tissues examined by real-time fluorescence quantitative PCR (qPCR), but the highest expression was found in the proboscis. This is the first report that antibacterial peptides have been found in C. tritonis, and it has been proved that Ct-20534 has antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, among which the activity of Staphylococcus aureus is most significantly inhibited, this suggests that the newly discovered antimicrobial peptides in C. tritonis may play an important role in the immune system and bacterial resistance of C. tritonis. This study presents the discovery of a newly identified antibacterial peptide from C. tritonis, with its structural properties fully characterized and potent antibacterial activity confirmed. The results provide essential fundamental data for the development of preventive and therapeutic measures against aquatic animal diseases, which in turn can promote the sustainable and stable growth of the aquaculture industry and create economic benefits. Additionally, this research lays the foundation for future development of novel anti-infective drugs.

Transcriptome analysis of gene expression profiling from the deep sea in situ to the laboratory for the cold seep mussel Gigantidas haimaensis.

BACKGROUND: The deep-sea mussel Gigantidas haimaensis is a representative species from the Haima cold seep ecosystem in the South China Sea that establishes endosymbiosis with chemotrophic bacteria. During long-term evolution, G. haimaensis has adapted well to the local environment of cold seeps. Until now, adaptive mechanisms responding to environmental stresses have remained poorly understood. RESULTS: In this study, transcriptomic analysis was performed for muscle tissue of G. haimaensis in the in situ environment (MH) and laboratory environment for 0 h (M0), 3 h (M3) and 9 h (M9), and 187,368 transcript sequences and 22,924 annotated differentially expressed genes (DEGs) were generated. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, these DEGs were enriched with a broad spectrum of biological processes and pathways, including those associated with antioxidants, apoptosis, chaperones, immunity and metabolism. Among these significantly enriched pathways, protein processing in the endoplasmic reticulum and metabolism were the most affected metabolic pathways. These results may imply that G. haimaensis struggles to support the life response to environmental change by changing gene expression profiles. CONCLUSION: The present study provides a better understanding of the biological responses and survival strategies of the mussel G. haimaensis from deep sea in situ to the laboratory environment.

Giant Triton Snail Charonia tritonis Macrophage-Expressed Gene 1 Protein Ct-Mpeg1: Molecular Identification, Expression Analysis, and Antimicrobial Activity.

Macrophage-expressed gene 1 proteins (Mpeg1/Perforin-2 (PRF2)) are a family of pore-forming proteins (PFPs) which can form pores and destroy the cell membrane of invading pathogens. However, little information is available regarding the function of Mpeg1 in the giant triton snail Charonia tritonis. In this study, a homolog of Mpeg1 (Ct-Mpeg1) was identified in C. tritonis. The predicted protein of Ct-Mpeg1 contains several structural features known in Mpegs, including a membrane attack complex/perforin (MACPF) domain and single transmembrane region. The Ct-Mpeg1 gene was constitutively expressed in almost all tissues examined except in the proboscis, with the highest expression level observed in the mantle. As a typical pore-forming protein, Ct-Mpeg1 has antibacterial activities against Vibrio (including Vibrio alginolyticus and Vibrio parahaemolyticus). In addition, rCt-Mpeg1 challenge to V. alginolyticus represses the expression of most outer membrane protein synthesis-related genes and genes involved in the TCA cycle pathway, which will lead to reduced outer membrane protein synthesis and less energy capacity. This is the first report to characterize the macrophage-expressed gene 1 protein in C. tritonis, and these results suggest that macrophage-expressed gene 1 protein Ct-Mpeg1 is an important immune molecule of C. tritonis that is involved in the bacterial infection resistance of Vibrio, and this study may provide crucial basic data for the understanding of the innate immunity system of C. tritonis.

Molecular and Functional Characterization of a Novel Kunitz-Type Toxin-like Peptide in the Giant Triton Snail Charonia tritonis.

It has been reported that the giant triton snail (Charonia tritonis) inserts its large proboscis and then injects venom or acid saliva from its salivary gland into its prey, the crown-of-thorns starfish Acanthaster planci (COTS), paralyzing it. A full-length cDNA sequence of the C. tritonis Ct-kunitzin gene was obtained by RACE PCR based on a transcriptomic database constructed by our laboratory (data not published), which contains an open reading frame (ORF) sequence with a length of 384 bp including a 1-32aa Kunitz domain. The Ct-kunitzin peptide was synthesized by solid-phase polypeptide methods according to its conserved amino acid sequence, with a molecular weight of 3746.0 as well as two disulfide bonds. Renatured Ct-kunitzin was injected into mice ventricles to evaluate its potential function. Compared with the normal control group (physiological saline), the spontaneous locomotor activity of the Ct-kunitzin group decreased significantly. There was a significant effect on Ct-kunitzin on mice grip strength in the grip strength test. In addition, Ct-kunitzin exhibited remarkable biological activity in suppressing pain in the pain thresholds test. There were no significant differences between the Ct-kunitzin group and the normal control group in terms of various hematological indexes and histopathological observations. When tested in COTS, the most significant histological change was the destruction, disorganization, and significant reduction in the amount of COTS tube feet tissues. Altogether, the potential paralyzing effect on mice suggests that Ct-kunitzin is a possible agent for novel drug development.

Molecular and Functional Characterization of a Short-Type Peptidoglycan Recognition Protein, Ct-PGRP-S1 in the Giant Triton Snail Charonia tritonis.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the potential function of PGRPs in the giant triton snail Charonia tritonis. In this study, a short-type PGRP gene (termed Ct-PGRP-S1) was identified in C. tritonis. Ct-PGRP-S1 was predicted to contain several structural features known in PGRPs, including a typical PGRP domain (Amidase_2) and Src homology-3 (SH3) domain. The Ct-PGRP-S1 gene was constitutively expressed in all tissues examined except in proboscis, with the highest expression level observed in the liver. As a typical PRR, Ct-PGRP-S1 has an ability to degrade peptidoglycan (PGN) and was proven to have non-Zn2+-dependent amidase activity and antibacterial activity against Vibrioalginolyticus and Staphylococcus aureus. It is the first report to reveal the peptidoglycan recognition protein in C. tritonis, and these results suggest that peptidoglycan recognition protein Ct-PGRP-S1 is an important effector of C. tritonis that modulates bacterial infection resistance of V. alginolyticus and S. aureus, and this study may provide crucial basic data for the understanding of an innate immunity system of C. tritonis.

Corrigendum: Effects of scale worm parasitism on interactions between the symbiotic gill microbiome and gene regulation in deep sea mussel hosts.

[This corrects the article DOI: 10.3389/fmicb.2022.940766.].

Effects of scale worm parasitism on interactions between the symbiotic gill microbiome and gene regulation in deep sea mussel hosts.

Diverse adaptations to the challenging deep sea environment are expected to be found across all deep sea organisms. Scale worms Branchipolynoe pettiboneae are believed to adapt to the deep sea environment by parasitizing deep sea mussels; this biotic interaction is one of most known in the deep sea chemosynthetic ecosystem. However, the mechanisms underlying the effects of scale worm parasitism on hosts are unclear. Previous studies have revealed that the microbiota plays an important role in host adaptability. Here, we compared gill-microbiota, gene expression and host-microorganism interactions in a group of deep sea mussels (Gigantidas haimaensis) parasitized by scale worm (PA group) and a no parasitic control group (NPA group). The symbiotic microorganism diversity of the PA group significantly decreased than NPA group, while the relative abundance of chemoautotrophic symbiotic bacteria that provide the host with organic carbon compounds significantly increased in PA. Interestingly, RNA-seq revealed that G. haimaensis hosts responded to B. pettiboneaei parasitism through significant upregulation of protein and lipid anabolism related genes, and that this parasitism may enhance host mussel nutrient anabolism but inhibit the host's ability to absorb nutrients, thus potentially helping the parasite obtain nutrients from the host. In an integrated analysis of the interactions between changes in the microbiota and host gene dysregulation, we found an agreement between the microbiota and transcriptomic responses to B. pettiboneaei parasitism. Together, our findings provide new insights into the effects of parasite scale worms on changes in symbiotic bacteria and gene expression in deep sea mussel hosts. We explored the potential role of host-microorganism interactions between scale worms and deep sea mussels, and revealed the mechanisms through which scale worm parasitism affects hosts in deep sea chemosynthetic ecosystem.

Proteome and Transcriptome Analysis of Gonads Reveals Intersex in Gigantidas haimaensis.

Sex has proven to be one of the most intriguing areas of research across evolution, development, and ecology. Intersex or sex change occurs frequently in molluscs. The deep-sea mussel Gigantidas haimaensis often dominates within Haima cold seep ecosystems, but details of their reproduction remain unknown. Herein, we conducted a combined proteomic and transcriptomic analysis of G. haimaensis gonads to provide a systematic understanding of sexual development in deep-sea bivalves. A total of 2,452 out of 42,238 genes (5.81%) and 288 out of 7,089 proteins (4.06%) were significantly differentially expressed between ovaries and testes with a false discovery rate (FDR) <0.05. Candidate genes involved in sexual development were identified; among 12 differentially expressed genes between sexes, four ovary-biased genes (ß-catenin, fem-1, forkhead box L2 and membrane progestin receptor α) were expressed significantly higher in males than females. Combining histological characteristics, we speculate that the males maybe intersex undergoing sex change, and implied that these genes may be involved in the process of male testis converting into female gonads in G. haimaensis. The results suggest that this adaptation may be based on local environmental factors, sedentary lifestyles, and patchy distribution, and sex change may facilitate adaptation to a changing environment and expansion of the population. The findings provide a valuable genetic resource to better understand the mechanisms of sex change and survival strategies in deep-sea bivalves.

Molecular cloning and functional characterization of KCNQ1 in shell biomineralisation of pearl oyster Pinctada fucata martensii.

KCNQ1, a voltage-gated potassium ion channel, plays an important role in various physiological processes, including osteoblast differentiation in higher animals. However, its function in lower invertebrates such as marine shellfish remains poorly understood. Pearl oysters, such as P. fucata martensii, are ideal for studying biomineralisation. In this study, a full-length cDNA of KCNQ1 from P. fucata martensii (PfKCNQ1) was obtained, and its function in shell formation was investigated. The full-length 3945 bp cDNA of PfKCNQ1 included an open reading frame (ORF) of 1944 bp encoding a polypeptide of 647 amino acids. Multiple sequence alignment revealed high homology with KCNQ1 from other species, with six transmembrane domains (S1 - S6) and a pore (P) region. Expression pattern analysis showed that PfKCNQ1 was expressed in all tested tissues, with highest expression in mantle and heart, and shell notching induced PfKCNQ1 expression. Silencing PfKCNQ1 expression inhibited PfKCNQ1 expression and downregulated four biomineralisation-related genes (Shematrin, Pif80, N16 and MSI60). Disordered crystals or "hollows" were visible in the shell ultrastructure by scanning electron microscopy following PfKCNQ1 knockdown. The results suggested that PfKCNQ1 may participate in or regulate biomineralisation and shell formation in pearl oyster.

The role of Smad1/5 in mantle immunity of the pearl oyster Pinctada fucata martensii.

The Smad protein family is an important medium for transducing BMP-Smads signals, and which have been proved that their important role in regulating shell biomineralization in Pinctada fucata martensii in our previous study. The members of TGF-ß superfamily were involved in innate immunity in vertebrates and invertebrates, and Smad regulatory networks construct a balanced immune system. However, little is known about the role of Smad1/5 in immunity in P. f. martensii. The present study shows that the tissue distribution and the expression profiles of Smad1/5 at developmental stages suggested its wide distribution and crucial role in development at embryonic stages other than larval stage; the increased expression of bone morphogenetic proteins 2 (BMP2), Smad4, Smad1/5 and MSX mRNAs at mantle tissue after LPS and Poly (I:C) challenged implied the potential immune role of Smad1/5 and BMP2-Smad signals to defense against bacterial and virus infections; the reduced expression of immune gene nuclear factor kappa-B (NF-κB), matrix metalloproteinase (MMP), interleukin 17 (IL-17), CuZn-superoxide dismutase (CuZn-SOD), tissue inhibitors of metalloproteinase (TIMP) and lipopolysaccharide-induced TNF-α factor (LITAF) mRNA following knockdown of Smad1/5 indicated that Smad1/5 can regulate their expression via BMP2-Smads pathway in the immunity process; the up-regulated expression of Smad1/5 and BMP2-Smad signals genes, and immune genes during wound healing indicated that Smad1/5 and BMP2-Smad signals genes may be involved in wound healing collaborated with immune genes via a different and complex Smads signaling pathway. These results indicated Smad1/5 could regulate innate immunity via BMP2-Smads signal pathway, and which provided new insights into the relationship between BMP2-Smads signal pathway and mantle immunity.

Comparative Transcriptomic and Expression Profiles Between the Foot Muscle and Mantle Tissues in the Giant Triton Snail Charonia tritonis.

The giant triton snail (Charonia tritonis), an endangered gastropod species of ecological and economic importance, is widely distributed in coral reef ecosystems of the Indo-West Pacific region and the tropical waters of the South China Sea. Limited research on molecular mechanisms can be conducted because the complete genomic information on this species is unavailable. Hence, we performed transcriptome sequencing of the C. tritonis foot muscle and mantle using the Illumina HiSeq sequencing platform. In 109,722 unigenes, we detected 7,994 (3,196 up-regulated and 4,798 down-regulated) differentially expressed genes (DEGs) from the C. tritonis foot muscle and mantle transcriptomes. These DEGs will provide valuable resources to improve the understanding of molecular mechanisms involved in biomineralization of C. tritonis. In the Gene Ontology (GO) database, DEGs were clustered into three main categories (biological processes, molecular functions, and cellular components) and were involved in 50 functional subcategories. The top 20 GO terms in the molecular function category included sulfotransferase activity, transferring sulfur-containing groups, and calcium ion binding, which are terms considered to be related to biomineralization. In KEGG classifications, transcriptomic DEGs were mainly enriched in glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate, and sulfur metabolism pathway, which may be related to biomineralization. The results of qPCR showed that three of the eight genes examined were significantly up-regulated in the mantle. The phylogenetic tree of BMP1 suggested a significant divergence between homologous genes in C. tritonis. Our results improve the understanding of biomineralization in C. tritonis and provide fundamental transcriptome information to study other molecular mechanisms such as reproduction.

PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome.

BACKGROUND: The pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms. RESULTS: Herein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment. CONCLUSIONS: Our results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.

The role of a new insulin-like peptide in the pearl oyster Pinctada fucata martensii.

Pinctada fucata martensii, is an economically important marine bivalve species cultured for seawater pearls. At present, we know little about the molecular mechanisms of the insulin signalling pathway in this oyster. Herein, we cloned and analysed an insulin-like peptide (PfILP) and its signalling pathway-related genes. We detected their expression levels in different tissues and developmental stages. Recombinant PfILP protein was produced and found to significantly increase primary mantle cell activity and induce the expression of the proliferating cell nuclear antigen (PCNA) gene. PfILP could also regulate the 293T cell cycle by stimulating the S phase and inhibiting the G1 and G2 phases. Recombinant PfILP protein induced the expression of its signalling pathway-related genes in mantle cells. In vitro co-immunoprecipitation analysis showed that PfILP interacts with PfIRR. PfILP activated expression of the pfIRR protein, and also activated the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways by stimulating phosphorylation of MAPK and AKT. Further analysis showed that PfILP up-regulated glycogen synthesis-related genes glycogen synthase kinase-3 beta (GSK-3ß), protein phosphatase 1 (PP1) and glucokinase (GK) at the mRNA level, as well as the expression of the PP1 protein, and phosphorylation of GSK-3ß. These results confirmed the presence of a conserved insulin-like signalling pathway in pearl oyster that is involved in cell activity, glycogen metabolism, and other physiological processes.

PfSMAD1/5 Can Interact with PfSMAD4 to Inhibit PfMSX to Regulate Shell Biomineralization in Pinctada fucata martensii.

The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein-protein action form and plays the role of transcription repressor.

Comparative transcriptomic and proteomic analysis of yellow shell and black shell pearl oysters, Pinctada fucata martensii.

BACKGROUND: The pearl oyster Pinctada fucata martensii (Pfu.), widely cultured in the South China Sea, is a precious source of sea pearls and calcifying materials. A yellow shell variety of Pfu. was obtained after years of artificial breeding. To identify differentially expressed genes between yellow shell and normal black shell pearl oysters, we performed transcriptomic sequencing and proteomic analyses using mantle edge tissues. RESULTS: A total of 56,969 unigenes were obtained from transcriptomic, of which 21,610 were annotated, including 385 annotated significant up-regulated genes and 227 significant down-regulated genes in yellow shell oysters (| log2 (fold change) | ≥2 and false discovery rate < 0.001). Tyrosine metabolism, calcium signalling pathway, phototransduction, melanogenesis pathways and rhodopsin related Gene Ontology (GO) terms were enriched with significant differentially expressed genes (DEGs) in transcriptomic. Proteomic sequencing identified 1769 proteins, of which 51 were significantly differentially expressed in yellow shell oysters. Calmodulin, N66 matrix protein, nacre protein and Kazal-type serine protease inhibitor were up-regulated in yellow shell oysters at both mRNA and protein levels, while glycine-rich protein shematrin-2, mantle gene 4, and sulphide: quinone oxidoreductase were down-regulated at two omics levels. Particularly, calmodulin, nacre protein N16.3, mantle gene 4, sulphide: quinone oxidoreductase, tyrosinase-like protein 3, cytochrome P450 3A were confirmed by quantitative real-time PCR. Yellow shell oysters possessed higher total carotenoid content (TCC) compared than black shell oyster based on spectrophotography. CONCLUSIONS: The yellow phenotype of pearl oysters, characterised by higher total carotenoids content, may reflect differences in retinal and rhodopsin metabolism, melanogenesis, calcium signalling pathway and biomineralisation. These results provide insights for exploring the relationships between calcium regulation, biomineralisation and yellow shell colour pigmentation.

Transcriptome analysis of mantle tissues reveals potential biomineralization-related genes in Tectus pyramis Born.

The marine mollusk Tectus pyramis is a valuable shellfish primarily distributed in the tropical waters of the South China Sea, as well as in the Indo-Pacific Ocean and areas near the southern portion of the Japanese Peninsula. Despite major economic interest in this mollusk, limited genomic resources are available for this species, which has prevented studies of the molecular mechanism, such as biomineralization. Here, we report the first comprehensive transcript dataset of T. pyramis mantle tissue. From a total of 16,801,141 reads, 173,671 unique transcripts were assembled, which provides new genomic resources for the understanding of biomineralization in T. pyramis. The most abundant unique sequences of the top 30 most highly expressed genes were annotated as shematrin, while other highly expressed genes included glycine-rich protein and shematrin-1. Based on transcriptome annotation and Gene Ontology classification, 130 biomineralization-related genes were found including members of the BMP (bone morphogenetic proteins), calmodulin, perlucin, and shematrin families, as well as mantle genes, nacrein, and MSI60. The results of qPCR showed that 14 of 24 examined genes were highly expressed in the mantle. A phylogenetic tree of BMP, perlucin, shematrin proteins revealed conservation of their structure and functions and indicated that some members participated in biomineralization in T. pyramis. Taken together, the results presented herein will be useful in studies of molecular mechanisms and pathways of biomineralization in T. pyramis, as well as provide new insight into the mechanisms of biomineralization in gastropods.

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

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.

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

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.

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, ß-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 ß-catenin may inhibit sry and activate 5-HT receptor and vitellogenin to maintain female development. High expression of sry may inhibit foxl2 and ß-catenin and activate dmrt2, fem-1, sfp2, sa6, Amy-1, APCP4, and PLK to maintain male function. High expression of sry, foxl2, and ß-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.

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 6 h, 48 h and 48 h, 72 h, 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.