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.

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 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.

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 cloning, characterization, and expression of two TNFRs from the pearl oyster Pinctada fucata martensii.

Proteins in the tumor necrosis factor receptor (TNFR) superfamily play significant roles in many physiological and pathological events, such as inflammation, apoptosis, autoimmunity, and organogenesis. Here, two TNFR gene homologs (PmTNFR1 and PmTNFR5) were identified in the pearl oyster Pinctada fucata martensii. The predicted PmTNFR1 and PmTNFR5 protein sequences were 406 and 533 amino acids long, respectively, and both possessed motifs characteristic of the TNFR family, including a TNFR homology domain (CRD), a transmembrane domain (TM), and death domains. However, the predicted amino acid sequences of PmTNFR1 and PmTNFR5 had low identity (~16-23%) with sequences of vertebrate TNFR family proteins. Furthermore, PmTNFR5 had a death domain at the C-terminal, indicating that this protein may be a novel member of the TNFR superfamily. Constitutive PmTNFR1 and PmTNFR5 mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the hepatopancreas and gill. The gene expression levels of PmTNFR1 and PmTNFR5, as well as those of downstream signaling molecules related to the NF-κB pathway (RIP, TRAF2, TRAF3, IKK, and NF-κB), were quantified in the gill after LPS challenge and in the hemocytes after nucleus insertion surgery using real-time PCR (qRT-PCR). We found that all genes were significantly upregulated at 6 h and 12 h post-injection, as well as at 15 d post-insertion. We used RNAi to inhibit the expression of the PmTNFR1 and PmTNFR5 genes. We then quantified the expression levels of PmTNFR1 and PmTNFR5, as well as downstream genes, using qRT-PCR. We found that RNAi inhibition of PmTNFR1 and PmTNFR5 downregulated the downstream genes (RIP, TRAF2, TRAF3, IKK, and NF-κB). Therefore, our results suggested that PmTNFR1 and PmTNFR5 mediate the NF-κB signaling pathway, and are closely related to immune defense, particularly allograft immunity, in the pearl oyster P. fucata martensii.

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.

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.

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.].