Antibiofilm activity of Morganella morganii JB8F and Pseudomonas fluorescens JB3B compound to control single and multi-species of aquaculture pathogens.

BACKGROUND: Indonesia is a country that uses half or more aquatic foods as protein intake. The increased production in aquaculture industries might cause several problems, such as bacterial disease resulting in mass mortality and economic losses. Antibiotics are no longer effective because aquaculture pathogens can form biofilm. Biofilm is a microbial community that aggregates and firmly attaches to living or non-living surfaces. Biofilm formation can be caused by environmental stress, the presence of antibiotics, and limited nutrients. Therefore, it is important to explore antibiofilm to inhibit biofilm formation and/or eradicate mature biofilm. Phyllosphere bacteria can produce bioactive compounds for antimicrobial, antibiofilm, and anti-quorum sensing. Three aquaculture pathogens were used in this study, such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. RESULTS: Pseudomonas fluorescens JB3B and Morganella morganii JB8F extracts could disrupt single and multi-species biofilms. Both extracts could inhibit single biofilm formation from one to seven days of incubation time. We confirmed the destruction activity on multi-species biofilm using light microscope and scanning electron microscope. Using GC-MS analysis, indole was the most active fraction of the P. fluorescens JB3B extracts and octacosane from the M. morganii JB8F extract. We also conducted a toxicity test using brine shrimp lethality assay on P. fluorescens JB3B and M. morganii JB8F extracts. P. fluorescens JB3B, M. morganii JB8F, and a mixture of both extracts were confirmed non-toxic according to the LC50 value of the brine shrimp lethality test. CONCLUSIONS: P. fluorescens JB3B and M. morganii JB8F phyllosphere extracts had antibiofilm activity to inhibit single biofilm and disrupt single and multi-species biofilm of aquaculture pathogens. Both extracts could inhibit single species biofilm until seven days of incubation. Bioactive compounds that might contribute to antibiofilm properties were found in both extracts, such as indole and phenol. P. fluorescens JB3B, M. morganii JB8F extracts, and mixture of both extracts were non-toxic against Artemia salina.

High temperatures increase the virulence of Vibrio bacteria towards their coral host and competing bacteria via type VI secretion systems.

The bacterial pathogen Vibrio coralliilyticus induces severe coral diseases in warming oceans. A study in PLOS Biology reveals that high temperatures activate 2 type VI secretion systems in V. coralliilyticus, enhancing pathogenicity by deploying toxic effectors against competing bacteria and coral cells.

PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus).

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

Prospection of strains of Bacillus sporogenes in the digestive tract of native crustaceans and characterization of the probiotic potential.

The cultivation of marine shrimp is one of the fastest growing activities in the world. However, the emergence of diseases has resulted in a decrease in production and losses for the sector. Probiotics emerged as an option to the use of antibiotics to control these pathogens. The efficiency of applying this technology depends on the characteristics of the bacterial agents and their bioavailability in the shrimp intestine. The objective is to evaluate the viability and efficiency of bacteria isolated from the digestive tract of healthy crustaceans as probiotic agents in the cultivation of shrimp Litopenaeus vannamei. Eighteen strains of the genus Bacillus belonging to the following species were tested: Bacillus sp., B. cereus, B. thuringiensis, B. circulans, B. megaterium, B. subtilis and B. agaridevorans. Bacterial isolates were subjected to characterization as potential probiotics. The test results were considered satisfactory; thus, the tested strains have potential for use as probiotics in shrimp culture. Treatments that used of the genus Bacillus had reduced growth of the genus Vibrio after infection, both in the intestinal contents and in the intestine. With the results obtained, it can be suggested that further research be carried out on the probiotic potential of Bacillus sp.

Bacterial recognition and inhibition activities of an LRR-only protein in the Chinese mitten crab, Eriocheir sinensis.

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20 ‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Bioemulsifier from sponge-associated bacteria reduces staphylococcal biofilm.

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Characteristics and functional analysis of a novel mannose receptor in Penaeus vannamei.

The mannose receptor (MR) plays a key role in the innate immune system as a pattern recognition receptor. Here, a novel type of mannose receptor, named PvMR2, was identified from Penaeus vannamei (P. vannamei). The PvMR2 coding sequence (CDS) obtained was 988 base pairs in length, encoding a protein consisting of 328 amino acids. This protein includes a signal peptide and two classical C-type lectin domains (CTLD). Quantitative real-time PCR showed that PvMR2 was distributed in all detected tissues, with the highest levels in the intestines and stomach. Following a bacterial challenge with Vibrio anguillarum (V. anguillarum), PvMR2 showed significant up-regulation in both the intestines and stomach of shrimp. To validate the function of PvMR2, recombinant proteins were extracted and purified using a His-tag. The resulting rPvMR2 demonstrated binding capability with lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner, affirming its binding affinity. The purified rPvMR2 demonstrated calcium-independent binding activity towards both Gram-positive bacteria (V. anguilliarum and Vibrio parahaemolyticus) and Gram-negative bacteria (Escherichia coli and Aeromonas Veronii). Antibacterial assays confirmed that rPvMR2 inhibits bacterial growth. Intestinal adhesion and adhesion inhibition experiments confirmed that the rPvMR2 can be used to reduce the adhesion capacity of harmful bacteria in the gut. Phagocytosis experiments have shown that rPvMR2 promotes phagocytosis in hemocytes and protects the host from external infection. Treatment with recombinant PvMR2 significantly bolstered bacterial clearance within the hemolymph and markedly augmented shrimp survival post-infection with V. anguillarum. These results suggest that PvMR2 has agglutination, growth inhibition, adhesion inhibition, clearance promotion, and phagocytosis effects on harmful bacteria, and plays a crucial role in the antimicrobial immune response of P. vannamei.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix.

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Effects of Vibrio harveyi infection on the biochemistry, histology and transcriptome in the hepatopancreas of ivory shell (Babylonia areolata).

The ivory shell (Babylonia areolata) is one of the most promising high quality marine products. However, ivory shell is susceptible to Vibrio harveyi infection during the culture period. In this study, we investigated the biochemical indicators, histological changes and transcriptomic response in the hepatopancreas of ivory shells from the PBS control group (PC) and infection group (A3) with 1 × 109 CFU/mL V. harveyi after 24 h. Results showed that compared to the PC group, biochemical indicators, including malondialdehyde (MDA), reactive oxygen species (ROS), acid phosphatase (ACP), and Caspase 3 (Casp-3) were significantly increased (p < 0.05) in A3 group after V. harveyi infection for 24 h. Compared with the PC group, the hepatopancreas of A3 group were seriously damaged, the columnar epithelial cells of the tissue were enlarged, the space of digestive cells was increased, and vacuolar cavities appeared. A total of 95,581 unigenes were obtained and 2949 (1787 up-regulated and 1162 down-regulated) differential expressed genes (DEGs) were identified in the A3 group. GO and KEGG enrichment analysis showed that DEGs were mainly enriched in immune system process (GO:0002376), antioxidant activity (GO:0016209), lysosome (ko04142), toll and IMD signaling pathway (ko04624), and etc. These biological functions and pathways are associated with immune and inflammatory responses and apoptosis. 12 DEGs were randomly selected for real-time quantitative PCR (RT-qPCR) validation, and the expression profiles of these DEGs were consistent with the transcriptome data, confirming the accuracy and reliability of the transcriptome results. In summary, V. harveyi infection of ivory shells inducing oxidative stress, leading to severe hepatopancreatic damage, stimulating glutathione production to neutralize excessive ROS, and stimulating antimicrobial peptides production to counteract the deleterious effects of bacterial infection, which in turn modifying the immune and inflammatory response, ultimately resulting in apoptosis. This study provided valuable information to explore the immune regulation mechanism after V. harveyi infection and established molecular basis to support the prevention of V. harveyi infection.

MiR-210, not let-7, regulates Akt gene expression against Vibrio splendidus infection in the sea cucumber Apostichopus japonicus.

The immune regulatory roles of microRNAs (miRNAs) have recently attracted considerable attention. Bioinformatics prediction revealed that both let-7 and miR-210 provide potential binding sites for the Akt (rac-alpha serine/threonine-protein kinase) gene sequence in the sea cucumber Apostichopus japonicus (termed AjAkt). In this study, we first used a dual-luciferase reporter assay and functional validation techniques to verify the interactions between these two miRNAs (let-7 and miR-210) and AjAkt, and then investigated the functions of the validated miRNA/mRNA pair as part of the innate immune response against Vibrio splendidus infection. We found that AjAkt interacts with miR-210 rather than let-7, and miR-210 negatively regulates the expression of AjAkt. From 8 to 48 h after infection with V. splendidus, opposite trends were observed in the expression levels of miR-210 and AjAkt (mRNA and protein) in coelomocytes, suggesting that the miR-210/AjAkt pair is involved in immune regulation during this period after infection. Both AjAkt silencing and miR-210 overexpression enhanced the phagocytic capacity and reduced the infectivity of A. japonicus after pathogen infection, suggesting that the miR-210/AjAkt pair may regulate the innate immune response of A. japonicus by altering phagocytic capacity. The findings of this study enrich our knowledge of the role of miRNA/mRNA pairs in immune regulation in sea cucumbers and provide insights into the molecular mechanisms of the innate immune response in marine echinoderms.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus.

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5'-untranslated region (UTR), and a 233 bp 3'-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37-335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Climate challenges for fish larvae: Interactive multi-stressor effects impair acclimation potential of Atlantic herring larvae.

Fish early life stages are particularly vulnerable and heavily affected by changing environmental factors. The interactive effects of multiple climate change-related stressors on fish larvae remain, however, largely underexplored. As rising temperatures can increase the abundance and virulence of bacteria, we investigated the combination of a spring heat wave and bacterial exposure on the development of Atlantic herring larvae (Clupea harengus). Eggs and larvae of Western Baltic Spring-spawners were reared at a normal and high temperature ramp and exposed to Vibrio alginolyticus and V. anguillarum, respectively. Subsequently, mRNA and miRNA transcriptomes, microbiota composition, growth and survival were assessed. Both high temperature and V. alginolyticus exposure induced a major downregulation of gene expression likely impeding larval cell proliferation. In contrast, interactive effects of elevated temperature and V. alginolyticus resulted in minimal gene expression changes, indicating an impaired plastic response, which may cause cellular damage reducing survival in later larval stages. The heat wave alone or in combination with V. alginolyticus induced a notable shift in miRNA expression leading to the down- but also upregulation of predicted target genes. Moreover, both increased temperature and the Vibrio exposures significantly altered the larval microbiota composition, with warming reducing microbial richness and diversity. The outcomes of this study highlight the high sensitivity of herring early life stages towards multiple climate change-related stressors. Our results indicate that interactive effects of rapidly changing environmental factors may exceed the larval stress threshold impairing essential acclimation responses, which may contribute to the ongoing recruitment decline of Western Baltic Spring-Spawning herring.

Characterization of quorum regulatory small RNAs in an emerging pathogen Vibrio fluvialis and their roles toward type VI secretion system VflT6SS2 modulation.

The type VI secretion system (T6SS) is essential for Gram-negative bacteria to antagonize a wide variety of prokaryotic and eukaryotic competitors and thus gain survival advantages. Two sets of T6SS have been found in Vibrio fluvialis, namely VflT6SS1 and VflT6SS2, among which VflT6SS2 is functionally expressed. The CqsA/LuxS-HapR quorum sensing (QS) system with CAI-1 and AI-2 as signal molecules can regulate VflT6SS2 by regulators LuxO and HapR, with LuxO repressing while HapR activating VflT6SS2. Quorum regulatory small RNAs (Qrr sRNAs) are Hfq-dependent trans-encoded sRNAs that control Vibrio quorum sensing. In V. fluvialis, Qrr sRNAs have not been characterized and their regulatory function is unknown. In this study, we first identified four Qrr sRNAs in V. fluvialis and demonstrated that these Qrr sRNAs are regulated by LuxO and involved in the modulation of VflT6SS2 function. On the one hand, Qrr sRNAs act on HapR, the activator of both the major and the auxiliary clusters of VflT6SS2, and then indirectly repress VflT6SS2. On the other hand, they directly repress VflT6SS2 by acting on tssB2 and tssD2_a, the first gene of the major cluster and the highly transcriptional one among the two units of the first auxiliary cluster, respectively. Our results give insights into the role of Qrr sRNAs in CAI-1/AI-2 based QS and VflT6SS2 modulation in V. fluvialis and further enhance understandings of the network between QS and T6SS regulation in Vibrio species.

Systemic immune responses do not affect significant immune responses in the skin.

Fish skin plays an important role in defending against pathogens in water, primarily through the secretion of skin mucus containing various immune-related factors. Local immune responses in the skin activate systemic immune responses by inflammatory cytokines. However, it remains unclear whether immune responses in the skin occur after systemic immune responses caused by pathogen invasion into the fish body. This study aimed to clarify the relationship between systemic immune responses and skin responses after intraperitoneal injection of formalin-killed cells (FKC) of Vibrio anguillarum. Although systemic inflammatory responses were observed in the spleen after injection, expression changes in the skin did not show significant differences. In contrast, expression of hemoglobin subunit genes significantly increased in the skin after FKC injection, suggesting that erythrocytes infiltrate extravascularly.

Preliminary investigation on the effect of Vibrio splendidus stimulation on the intestinal flora of Strongylocentrotus intermedius.

To better understand the effect of Vibrio splendidus infection on Strongylocentrotus intermedius, 16S rRNA sequencing was carried out to investigate the intestinal flora of S. intermedius stimulated by 0 CFU/mL (Con), 1.5 × 107 CFU/mL (Vib1) and 1.5 × 108 CFU/mL (Vib2) concentrations of V. splendidus. The results showed that there was significant difference in intestinal flora diversity between Con group and Vib1 group, but no significant difference between Con group and Vib2 group. However, there were significant differences in the composition of intestinal flora among all groups. Bacteroidota, Proteobacteria and Firmicutes were the dominant phylum in the Con group. The abundance of Bacteroidota and Firmicutes decreased and Proteobacteria increased in Vib1 and Vib2 groups. The relative abundance of the potential probiotic bacteria Muribaculaceae and Alloprevotella was significantly lower in the Vib1 and Vib2 groups. In addition, the opportunistic pathogen Desulfovibrio was found in Vib1 and Vib2 groups. It is evident that V. splendidus infection not only alters the composition of the microbial community in the intestinal tract of S. intermedius, but may also lead to the production of opportunistic pathogens, which could be potentially harmful to the health of S. intermedius. The results of this study provide a foundation for exploring the diseases caused by V. splendidus stimulation leading to an imbalance in the intestinal flora of S. intermedius, and contribute to our further understanding of the role of Vibrio on the health of S. intermedius.

The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

Immune and regulative characterization of complement-related gene cfhl5 in response to Vibrio harveyi challenge in Cynoglossus semilaevis.

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1ß (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole.

Characterization of Myxovirus resistance (Mx) gene from Chinese seabass Lateolabrax maculatus: Insights into the evolution and function of Mx genes.

Chinese seabass (Lateolabrax maculatus) stands out as one of the most sought-after and economically significant species in aquaculture within China. Diseases of L. maculatus occur frequently due to the degradation of the germplasm, the aggravation of environmental pollution of water, and the reproduction of pathogenic microorganisms, inflicting considerable economic losses on the Chinese seabass industry. The Myxovirus resistance (Mx) gene plays pivotal roles in the antiviral immune response ranging from mammals to fish. However, the function of the Mx gene in L. maculatus is still unknown. Firstly, the origin and evolutionary history of Mx proteins was elucidated in this study. Subsequently, an Mx gene from L. maculatus (designed as LmMxA gene) was identified, and its functions in combating antiviral and antibacterial threats were investigated. Remarkably, our findings suggested that while Mx group genes were present in chordates, DYN group genes were present in everything from single-celled animals to humans. Furthermore, our investigation revealed that the LmMxA mRNA level increased in the kidney, spleen and liver subsequent to Vibrio anguillarum and poly(I:C) challenged. Immunofluorescence analysis indicated that LmMxA is predominantly localization in the nucleus and the cytoplasm. Notably, the expression of MAVS, IFN1 and Mx1 increased when LmMxA was overexpression within the EPC cells. Moreover, through assessment via cytopathic effect (CPE), virus titer, and antibacterial activity, it becomes evident that LmMxA exerts a dual role in bolstering both antiviral and antibacterial immune responses. These compelling findings laid the foundation for further exploring the mechanism of LmMxA in response to innate immunity of L. maculatus.

Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major.

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.