Transcriptome analysis provides new insights into the immune response of Ruditapes philippinarum infected with Vibrio alginolyticus.

Manila clam (Ruditapes philippinarum) is a bivalve species with commercial value, but it is easily infected by pathogenic microorganisms in aquaculture, which restricts the shellfish industry. Notably, the impact of Vibrio alginolyticus on clam culture is obvious. In this study, RNA-seq was performed to analyze clam hepatopancreas tissue in 48 h (challenge group, G48h) and 96 h (challenge group, G96h) after infection with V. alginolyticus and 0 h after injection of PBS (control group, C). The results showed that a total of 1670 differentially expressed genes were detected in the G48h vs C group, and 1427 differentially expressed genes were detected in the G96h vs C group. In addition, KEGG analysis showed that DEGs were significantly enriched in pathways such as Lysosome and Mitophagy. Moreover, 15 immune related DEGs were selected for qRT-PCR analysis to verify the accuracy of RNA-seq, and the results showed that the expression level of DEGs was consistent with that of RNA-seq. Therefore, the results obtained in this study provides a preliminary understanding of the immune defense of R. philippinarum and molecular insights for genetic breeding of V. alginolyticus resistance in Manila clam.

Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin ß dependent nuclear translocation in Apostichopus japonicus.

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

Coral mucus as a reservoir of bacteriophages targeting Vibrio pathogens.

The increasing trend in sea surface temperature promotes the spread of Vibrio species, which are known to cause diseases in a wide range of marine organisms. Among these pathogens, Vibrio mediterranei has emerged as a significant threat, leading to bleaching in the coral species Oculina patagonica. Bacteriophages, or phages, are viruses that infect bacteria, thereby regulating microbial communities and playing a crucial role in the coral's defense against pathogens. However, our understanding of phages that infect V. mediterranei is limited. In this study, we identified two phage species capable of infecting V. mediterranei by utilizing a combination of cultivation and metagenomic approaches. These phages are low-abundance specialists within the coral mucus layer that exhibit rapid proliferation in the presence of their hosts, suggesting a potential role in coral defense. Additionally, one of these phages possesses a conserved domain of a leucine-rich repeat protein, similar to those harbored in the coral genome, that plays a key role in pathogen recognition, hinting at potential coral-phage coevolution. Furthermore, our research suggests that lytic Vibrio infections could trigger prophage induction, which may disseminate genetic elements, including virulence factors, in the coral mucus layer. Overall, our findings underscore the importance of historical coral-phage interactions as a form of coral immunity against invasive Vibrio pathogens.

Unravelling the main immune repertoire of Paracentrotus lividus following Vibrio anguillarum bath challenge.

Paracentrotus lividus is the most abundant echinoid species in the North East Atlantic Ocean and Mediterranean Sea. Although there is abundant genomic information of the species, there is no deep characterisation of the genes involved in the immune response. Here, a reference transcriptome of male and female coelomocytes was produced. The generated P. lividus transcriptome assembly has 203,511 transcripts, N50 transcript length of 1079 bp, and more than 90% estimated gene completeness in Eukaryota and Metazoa BUSCO databases, respectively. Differential gene expression analyses showed 54 and 55 up-regulated genes in P. lividus female and male coelomocyte tissues, respectively. These results suggest a similar immune gene repertoire between sexes. To examine the immune response, P. lividus was challenged with Vibrio anguillarum, one of the candidate pathogens for bald disease. Immune parameters were evaluated at cell and humoral levels, as well as the expression analysis of immune related genes at an early response stage. No differences were found at cellular and humoral levels with the exception of the increase of nitric oxide in perivisceral fluid of challenged animals. At the gene expression level, a total of 2721 genes were upregulated in challenged animals, 13.6 times higher expression than control group. Our analysis revealed that four major KEGG pathways were enriched in challenged animals: Autophagy (KEGG:04140), Endocytosis (KEGG:04144), Phagosome (KEGG:04145) and Protein processing in endoplasmic reticulum (KEGG:04141). Several toll-like receptors (TLR), scavenger receptors cysteine-rich (SRCR) or nucleotide-binding oligomerisation domain like receptors (NLR) were identified as major family genes for pathogen recognition and immune defence. This study provides a valuable transcriptomic resource and unfolds the molecular basis of immune response to V. anguillarum exposure. Overall, our findings contribute to the conservation effort of the P. lividus populations, as well as its sustainable exploitation in an aquaculture context.

Antagonistic activity of Phaeobacter piscinae against the emerging fish pathogen Vibrio crassostreae in aquaculture feed algae.

Aquaculture provides a rich resource of high-quality protein; however, the production is challenged by emerging pathogens such as Vibrio crassostreae. While probiotic bacteria have been proposed as a sustainable solution to reduce pathogen load in aquaculture, their application requires a comprehensive assessment across the aquaculture food chain. The purpose of this study was to determine the antagonistic effect of the potential probiotic bacterium Phaeobacter piscinae against the emerging fish pathogen V. crassostreae in aquaculture feed algae that can be an entry point for pathogens in fish and shellfish aquaculture. P. piscinae strain S26 produces the antibacterial compound tropodithietic acid (TDA). In a plate-based assay, P. piscinae S26 was equally to more effective than the well-studied Phaeobacter inhibens DSM17395 in its inhibition of the fish pathogens Vibrio anguillarum 90-11-286 and V. crassostreae DMC-1. When co-cultured with the microalgae Tetraselmis suecica and Isochrysis galbana, P. piscinae S26 reduced the maximum cell density of V. crassostreae DMC-1 by 2 log and 3-4 log fold, respectively. A TDA-deficient mutant of P. piscinae S26 inhibited V. crassostreae DMC-1 to a lesser extent than the wild type, suggesting that the antagonistic effect involves TDA and other factors. TDA is the prime antagonistic agent of the inhibition of V. anguillarum 90-11-286. Comparative genomics of V. anguillarum 90-11-286 and V. crassostreae DMC-1 revealed that V. crassostreae DMC-1 carries a greater arsenal of antibiotic resistance genes potentially contributing to the reduced effect of TDA. In conclusion, P. piscinae S26 is a promising new candidate for inhibition of emerging pathogens such as V. crassostreae DMC-1 in algal feed systems and could contribute to a more sustainable aquaculture industry.IMPORTANCEThe globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae. These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Microbial community and transcriptional responses to V. coralliilyticus stress in coral Favites halicora and Pocillopora damicornis holobiont.

Variability in coral hosts susceptibility to Vibrio coralliilyticus is well-documented; however, the comprehensive understanding of tolerance of response to pathogen among coral species is lacked. Herein, we investigated the microbial communities and transcriptome dynamics of two corals in response to Vibrio coralliilyticus. Favites halicora displayed greater resistance to Vibrio coralliilyticus challenge than Pocillopora damicornis. Furthermore, the relative abundances of Flavobacteriaceae, Vibrionacea, Rhodobacteraceae, and Roseobacteraceae increased significantly in Favites halicora following pathogen stress, whereas that of Akkermansiaceae increased significantly in Pocillopora damicornis, leading to bacterial community imbalance. In contrast to the previous results, pathogen infection did not have much effect on the community structures of Symbiodiniaceae and fungi, but led to a decrease in the density of Symbiodiniaceae. Transcriptome analysis indicated that Vibrio infection triggered a coral immune response, resulting in higher expression of immune-related genes, which appeared to have higher transcriptional plasticity in Favites halicora than in Pocillopora damicornis. Specifically, the upregulated genes of Favites halicora were predominantly involved in the apoptosis pathway, whereas Pocillopora damicornis were significantly enriched in the nucleotide excision repair and base excision repair pathways. These findings suggest that coral holobionts activate different mechanisms across species in response to pathogens through shifts in microbial communities and transcriptomes, which provides novel insight into assessing the future coral assemblages suffering from disease outbreaks.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection.

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

Gut microbiota dynamics interacting with gastrointestinal evacuation of Apostichopus japonicus: novel insights into promising strategies for environmental improvement.

As an important input of environmental micropollutants into aquaculture environment, feed is now considered to be a critical factor in shaping gastrointestinal evacuation characteristics of animals. We analyzed the gastrointestinal evacuation characteristics and gut bacteria of Apostichopus japonicus within 30 h after feeding in recirculating aquaculture system (RAS) and explored the evacuation mechanism interacting by bacteria. The Gauss model was the most precise gastrointestinal evacuation curve, and 80% of gastrointestinal evacuation time was 27.81 h after feeding. Linear discriminant analysis effect size analysis revealed that gut microbial abundance associated significantly with time (P < 0.05), and 42 biomarkers that could predict gastrointestinal evacuation were totally detected, such as Lutibacter and Vibrio. Biomarkers at 25 h after feeding were related to harmful bacteria. A dynamic response between gastrointestinal content ratio and gut microbial abundance was detected. Taken together, we could discharge sewage about 25 h after feeding and carry out the next round of feeding activities.

Comparative transcriptome analysis reveals potential regulatory mechanisms of genes and immune pathways following Vibrio harveyi infection in red drum (Sciaenops ocellatus).

Red drum (Sciaenops ocellatus), as an important economical marine fish, has been affected by various bacterial diseases in recent years. Vibrio harveyi cause fatal vibriosis in S. ocellatus, leading to massive mortality and causing significant setbacks in aquaculture. However, the regulatory mechanisms of S. ocellatus response to V. harveyi infection are poorly understood. In this regard, we performed transcriptomic analysis with head kidney tissues of S. ocellatus after V. harveyi infection from 12 h to 48 h to reveal genes, gene expression profiles, and pathways involved in immune and inflammation responses. Specifically, a total of 9,599, 5,728, and 7144 differentially expressed genes (DEGs) were identified after V. harveyi infection at 12 h, 24 h, and 48 h, respectively, and 1,848 shared DEGs have been identified from the above three comparison groups. Subsequent pathway analysis revealed that the shared DEGs following V. harveyi were involved in complement and coagulation cascades (C1R, C1QC, C3, C4, C5, C7, C8A, C8B, C8G, C9, CFB, CFH, and CFI), MAPK signaling pathway, chemokine signaling pathway (CCL19, CXCL8, CXCL12, CXCL14, CCR4, CCR7, and CXCR2), PPAR signaling pathway (PPAR-α, PPAR-γ and PPAR-ß), and TNF signaling pathway. Finally, the expression patterns of DEGs in head kidney tissues and S. ocellatus macrophages were validated by qRT-PCR, suggesting the reliability of RNA sequencing for gene expression analysis. This dynamic transcriptome analyses provided insights into gene expression regulation and immune related pathways involved in S. ocellatus after V. harveyi infection, and provides useful information for further study on the immune defense mechanisms in S. ocellatus as well as other teleost species.

Mucous cell histopathology and label-free quantitative proteomic analysis of skin mucus in fat greenling (Hexagrammos otakii) infected with Vibrio harveyi.

Hexagrammos otakii is favored by consumers and aquaculture practitioners because of its strong adaptability and fast growth. However, recently, frequent outbreaks of diseases in the breeding of H. otakii have led to significant economic losses, especially due to bacterial diseases, which limit the healthy breeding of H. otakii. As a luminescent Gram-negative bacterium, Vibrio harveyi is the main pathogenic bacteria of H. otakii. In this study, the histopathology and label-free quantitative proteomics analysis were performed to reveal the changes of skin mucus proteins in H. otakii after infection with V. harveyi. The histopathological changes in the skin of H. otakii showed that when the bacteria were injected into the epithelial cells, it caused an increase in the number of mucous cells and a certain degree of damage and deformation in skin. Moreover, the quantitative proteomics analysis revealed a total of 364 differentially expressed proteins (DEPs), and these DEPs were found to be involved in environmental information processing, metabolism, infectious diseases: bacteria, replication and repair. More importantly, the enrichment analysis of the DEPs revealed that these different proteins were mainly targeted immune-related pathways. After infection of bacteria, the host's immune ability will be weakened, causing V. harveyi to enter the organism more easily, resulting in increased mucus in H. otakii, which will eventually lead to a decline in its physical function. These results provided an insight into a series of physiological changes after the bacterial infection of fish at the proteomic level and basic data for further exploration of the potential mechanism of skin mucus. Taken together, the results indicated more opportunities for the future designs and discoveries of effective antibacterial vaccines and antibacterial drugs for H. otakii.

Characterization of caspase gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Vibrio anguillarum infection.

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii, we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6, and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii, especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii, we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida, the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii, thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes.

Effects of miR-722 on gene expression and alternative splicing in the liver of half-smooth tongue sole after infection with Vibrio anguillarum.

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Dietary Bacillus cereus LS2 protects juvenile sea cucumber Apostichopus japonicus against Vibrio splendidus infection.

This study aimed to investigate the effects of Bacillus cereus LS2 on the growth performance, innate immunity, intestinal microbiota, and disease resistance of sea cucumber Apostichopus japonicus. After feeding with LS2 for 30 days, results showed that dietary with LS2 had a significant improvement in the growth rate and immune parameters (including total coelomocytes counts, phagocytosis, respiratory burst, and immune-related enzymes) of juvenile sea cucumbers. Subsequently, transcriptome sequencing and qRT-PCR verification were performed to analyze the potential mechanism of LS2 diet and thus improve the immune response of A. japonicus. GO and KEGG pathway analysis indicated that LS2 can primarily activate the "Lectins" and "complement and coagulation cascades" pathways to modulate the innate immunity of the sea cucumbers. Furthermore, 16S rRNA sequencing was used to analyze the intestinal microbial composition of sea cucumbers after dietary with LS2. Results showed that Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were the most prevalent phyla in A. japonicus intestinal microbiota. The abundance of Actinobacteria (46.20%) and Bacteroidetes (12.80%) were significantly higher in the LS2 group, whereas the relative abundance of Proteobacteria (49.98%) and Firmicutes (14.97%) were higher in the control group. The LDA scores of Nocardiaceae and Rhodococcus were also the highest taxa after the dietary administration of LS2, indicating that Actinobacteria phylum played a pivotal role in the intestinal microbial function of A. japonicus. Overall, these results suggested that feeding with Bacillus LS2 may be beneficial for A. japonicus farming.

Pathogenicity and transcriptomic exploration of Vibrio fortis in Penaeus monodon.

In this study, a strain (recorded as Y6) was isolated from the biofloc pool, its DNA was extracted for 16S rDNA sequencing and compared in the NCBI database, and it was identified as Vibrio fortis. The V. fortis was activated, cultured, and artificially injected into Penaeus monodon to observe the symptoms and calculate the semi-lethal concentration (LC50). It was found that the symptoms of the red leg, an empty stomach, and enlarged hepatopancreas of P. monodon after infection with V. fortis. The LC50 was 4.00 × 107, 2.24 × 107, 1.82 × 107, 1.41 × 107, 7.52 × 106 and 3.31 × 106 CFU/mL at 16, 24, 32, 48, 128, and 144 hpi, respectively. The K-B disk method was used to detect the sensitivity of V. fortis to various antibiotic drugs. V. fortis resisted Ampicillin, Piperacillin, Cefazolin, Cephalothin and Cefoxitin. Highly sensitive to Polymyxin B, Tobramycin, Gentamicin, Cefepime, Cefoperazone and Streptomycin. To explore the molecular response mechanism of V. fortis infection in P. monodon, the hepatopancreas of P. monodon infected with V. fortis at 24 and 48 hpi by transcriptome sequencing, and a total of 347 DEGs were obtained (214 up-regulated DEGs and 133 down-regulated DEGs). In the KEGG pathway enrichment analysis of DEGs, significant changes were found in genes and signaling pathways related to immune system and substance metabolism, including NOD-like receptor signaling pathways, Toll and Imd signaling pathways, C-type lectin receptor signaling pathways and pyruvate metabolism. This study initially revealed the immune response of P. monodon to V. fortis infection from the molecular level and provided a reference for further understanding of the study and control of the vibriosis of shrimp.

Ajpacifastin-like is involved in the immune response of Apostichopus japonicus challenged by Vibrio splendidus.

Pacifastin proteins are previously found to regulate the phenoloxidase system in invertebrates and arthropods. In this study, the immune response that was regulated by Ajpacifastin-like in the sea cucumber Apostichopus japonicus was determined. RNA interference was used to knock down the expression of the Ajpacifastin-like gene in A. japonicus, followed by challenge with Vibrio splendidus, and the colony count showed that the survival of V. splendidus in the si-Ajpacifastin group increased 4.64-fold compared to that of the control group. The purified recombinant Ajpacifastin-like showed an inhibitory effect on the extracellular protease activity of the supernatant collected from the V. splendidus culture. Consequently, a comparative transcriptome analysis of the coelomocytes from the control group and the si-Ajpacifastin group was performed to explore the global regulatory effect of the Ajpacifastin-like. A total of 1486 differentially expressed genes (DEGs) were identified, including 745 upregulated genes and 741 downregulated genes. GO enrichment showed that the DEGs were mainly enriched in translation, cytosolic ribosomal subunit and structural constituent of ribosome. KEGG analysis showed that the DEGs were significantly enriched in the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor-kappa B signaling pathway and other immune-related pathways. Furthermore, real-time reverse transcriptase PCR was used to determine the RNA levels of six DEGs, i.e., cathepsinB, CYLD, caspase8, TRAF6, hsp90 and FADD, to verify the RNA-seq results. Overall, our results specified the immune response and pathways of A. japonicus in which Ajpacifastin-like was involved in.

A C-type lectin-like receptor CD302 in yellow drum (Nibea albiflora) functioning in antibacterial activity and innate immune signaling.

Molecular dissection of disease resistance against Vibrio harveyi infection in yellow drum at the genome-wide level uncovered a C-type lectin-like receptor cluster of differentiation CD302 (named as YdCD302) in our previous study. Here, the gene expression pattern of YdCD302 and its function in mediating the defense response to V. harveyi attack were investigated. Gene expression analysis demonstrated that YdCD302 was ubiquitously distributed in various tissues with the highest transcript abundance in liver. The YdCD302 protein exhibited agglutination and antibacterial activity against V. harveyi cells. Binding assay indicated that YdCD302 can physically interact with V. harveyi cells in a Ca2+-independent manner, and the interaction can activate reactive oxygen species (ROS) production in the bacterial cells to induce RecA/LexA-mediated cell death. After infection with V. harveyi, the expression of YdCD302 can be up-regulated significantly in the main immune organs of yellow drum and potentially further trigger the cytokines involved innate immunity. These findings provide insight into the genetic basis of the disease resistance trait in yellow drum and shed light on the functioning of the CD302 C-type lectin-like receptor in host-pathogen interactions. The molecular and functional characterization of YdCD302 is a significant step towards a better understanding of disease resistance mechanisms and the development of new strategies for disease control.

The TRAF gene family in turbot (Scophthalmus maximus): Identification, characterization, molecular evolution and expression patterns analysis.

Tumor necrosis factor receptor-associated factor (TRAF) is an important structural protein, which can bind to TNF receptors and participate in the regulation of TNF signaling pathway. Nonetheless, few studies have been conducted to investigate the systematic identification of TRAF gene family in teleost and role in innate immunity of turbot (Scophthalmus maximus). In this study, eight TRAF genes, namely SmTRAF2aa, SmTRAF2ab, SmTRAF2b, SmTRAF3, SmTRAF4a, SmTRAF5, SmTRAF6 and SmTRAF7, were identified and annotated in turbot by using bioinformatics methods. Analysis of the phylogenetic, syntenic and molecular evolution demonstrated that all SmTRAF members were evolutionarily conserved in teleost. Domain analysis showed all SmTRAF proteins contained a typical conserved N-terminal RING finger domain. Most SmTRAF proteins contained a MATH domain at the C-terminal, while SmTRAF7 contains seven duplicate WD40 domains. In addition, quantitative real-time PCR was performed to detect the expression patterns of SmTRAFs in tissues from healthy and Vibrio anguillarum infected turbots. The results indicated SmTRAFs had diverse tissue expression patterns and the expression of TRAF gene changed significantly after V. anguillarum infection. This study provided a basis for understanding the roles of TRAFs in the innate immune response of turbot.

A novel perlucin-like protein (PLP) protects Litopenaeus vannamei against Vibrio harveyi infection.

C-type lectins (CTLs), as pattern recognition receptors (PRRs), play an important role in the innate immunity of Litopenaeus vannamei. In this study, a novel CTL, named perlucin-like protein (PLP), was identified from L. vannamei, which shared homology sequences of PLP from Penaeus monodon. PLP from L. vannamei was expressed in the hepatopancreas, eyestalk, muscle and brain and could be activated in the tissues (hepatopancreas, muscle, gill and intestine) after infection with the pathogen Vibrio harveyi. Bacteria (Vibrio alginolyticus, V. parahaemolyticus, V. harveyi, Streptococcus agalactiae and Bacillus subtilis) could be bound and agglutinated by the PLP recombinant protein in a Ca2+-dependent manner. Moreover, PLP could stabilise the expression of the immune-related genes (ALF, SOD, HSP70, Toll4 and IMD) and apoptosis gene (Caspase2). The RNAi of PLP could remarkably affect the expression of antioxidant gene, antimicrobial peptide genes, other CTLs, apoptosis genes, Toll signaling pathways, and IMD signaling pathways. Moreover, PLP reduced the bacterial load in the hepatopancreas. These results suggested that PLP was involved in the innate immune response against V. harveyi infection by recognising bacterial pathogens and activating the expression of immune-related and apoptosis genes.