Monascus purpureus M-32 fermented soybean meal improves the growth, immunity parameters, intestinal morphology, disease resistance, intestinal microbiota and metabolome in Pacific white shrimp (Litopenaeus vannamei).

This study was conducted to evaluate the effects of Monascus purpureus M-32 fermented soybean meal (MFSM) on growth, immunity, intestinal morphology, intestinal microbiota, and intestinal metabolome of Pacific white shrimp (Litopenaeus vannamei). Four groups of diets were formulated, including control group (30% fish meal and 30% soybean meal [SBM] included in the basal diet) and three experimental groups which MFSM replaced 20% (MFSM20), 40% (MFSM40), and 60% (MFSM60) of SBM in control group, respectively. Results showed that the soluble proteins larger than 49 kDa in MFSM were almost completely degraded. Meanwhile, the crude protein, acid-soluble protein, and amino acid in MFSM were increased. The results of shrimp culture experiment showed that the replacement of SBM with MFSM decreased FCR (P < 0.001) and content of malondialdehyde (P = 0.007) in the experimental groups, and increased weight gain rate (P = 0.006), specific growth rate (P = 0.002), survival rate (P = 0.005), intestinal villus height (P < 0.001), myenteric thickness (P = 0.002), the activities of superoxide dismutase (P = 0.002), and lysozyme (P = 0.006) in experimental groups, as well as increased content of calcium (Ca2+) and phosphorus (PO43-) in blood and muscle, and enhanced resistance to Vibrio parahaemolyticus infection. The gut microbiota of MFSM groups was significantly different from that of the control group, and the abundance of Actinobacteria and Verrucomicrobia increased significantly in the MFSM60 group, whereas Proteobacteria and Firmicutes decreased. Compared with the control group, there were significant changes in the levels of several intestinal metabolites in the MFSM60 group, including leukotriene C5, prostaglandin A1, taurochenodeoxycholic acid, carnosine, and itaconic acid. The fermentation of SBM by the strain M. purpureus M-32 has the potential to enhance the nutritional quality of SBM, promote the growth of L. vannamei, boost immune response, improve intestinal morphology and microbiota composition, as well as influence intestinal metabolites.

Comparative transcriptome analysis explored the molecular mechanisms of a luxR-type regulator regulating intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila is not a traditional intracellular bacterium. However, previous studies revealed that pathogenic A. hydrophila B11 could temporarily survive for at least 24 h in fish phagocytes, and the regulation of intracellular survival in bacteria was associated with regulators of the LuxR-type. The mechanisms of luxR08110 on the A. hydrophila's survival in macrophages were investigated using comprehensive transcriptome analysis and biological phenotype analysis in this study. The results showed that after luxR08110 was silenced, the intracellular survival ability of bacteria was significantly diminished. Comparative transcriptome analysis revealed that luxR08110 was a critical regulator of A. hydrophila, which regulated the expression of over 1200 genes, involving in bacterial flagellar assembly and chemotaxis, ribosome, sulphur metabolism, glycerolipid metabolism, and other mechanisms. Further studies confirmed that after the inhibition of expression of luxR08110, the motility, chemotaxis and adhesion of A. hydrophila significantly decreased. Moreover, compared with the wild-type strain, the survival rates of silencing strain were all considerably reduced under both H2O2 and low pH stress conditions. According to both transcriptome analysis and phenotypic tests, the luxR08110 of A. hydrophila could act as global regulator in bacteria intracellular survival. This regulator regulated intracellular survival of A. hydrophila mainly through two ways. One way is to regulate bacterial flagellar synthesis and further affects the motility, chemotaxis and adhesion of bacteria. The other way is to regulate sulphur and glycerolipid metabolisms, thus affecting bacterial energy production and the ability to resist environmental stress.

The involvement of the T6SS vgrG gene in the pathogenicity of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida, the causative agent of white spot disease of large yellow croaker, has caused serious economic losses to the aquaculture industry. The type VI secretion system (T6SS) is a significant virulence system widely distributed among Gram-negative bacteria. VgrG, a structural and core component of T6SS, is crucial to the function of T6SS. To explore the biological profiles mediated by vgrG gene and its effects on the pathogenicity of P. plecoglossicida, the vgrG gene deletion (ΔvgrG) strain and complementary (C-ΔvgrG) strain were constructed and the differences in pathogenicity and virulence-related characteristics between different strains were analysed. The results showed that vgrG gene deletion significantly affected the virulence-related characteristics of P. plecoglossicida, including chemotaxis, adhesion, and biofilm formation. In addition, the LD50 of ΔvgrG strain was nearly 50-fold higher than that of the NZBD9 strain. Transcriptome data analysis suggested that the vgrG gene may affect the virulence of P. plecoglossicida by regulating the quorum sensing pathway to inhibit the secretion of virulence factors and affect biofilm formation. Besides, deletion of the vgrG gene may reduce bacterial pathogenicity by affecting bacterial signal transduction processes and the ability to adapt to chemotactic substances.

Pseudomonas plecoglossicida fliP gene affects the immune response of Epinephelus fuscoguttatus ♀×Epinephelus lanceolatus ♂ to infection.

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease in a variety of teleosts. The protein encoded by fliP gene is involved in the assembly of bacterial flagella, which plays a vital role in bacterial pathogenicity. However, the roles of the fliP gene on the host immune response remain unclear. Here, we compared the pathogenicity of fliP gene-deleted (ΔfliP) strain, fliP gene-complemented (C-ΔfliP) strain and wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), and explored the impacts of fliP gene on the immune response of hybrid grouper to P. plecoglossicida infection by using RNA-seq. In this study, the grouper in the ΔfliP strain-infected group had a 30% higher survival rate than those in the NZBD9 strain-infected group. In addition, the deletion of fliP gene decreased bacterial load in the spleen, intestine, liver as well as head kidney of hybrid grouper and the tissues damage were weakened. Moreover, the infection of hybrid grouper spleen by the ΔfliP strain induced 1,189 differential expression genes compared with the counterpart infected by NZBD9 strain. KEGG enrichment analysis showed that 9 immune-related pathways, 5 signal transduction pathways, and 3 signaling molecules and interaction pathways were significantly enriched. qRT-PCR analysis revealed that the ΔfliP strain mainly up-regulated the expression of inflammation related genes (IL-6, IL-12, IL-1ß, IL-10, CXCL8, CXCL10) and immune regulation related genes (TLR2, P65, MyD88, P85, AKT), but down-regulated the expression of cell death related genes (FoxO1, Bim, PLK2 and LDHA) during infection. Based on the above results, fliP gene contributed to the pathogenicity of P. plecoglossicida to hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂), deletion of fliP gene promoted the inflammation and immune response of hybrid grouper to P. plecoglossicida infection, which accelerating host clearance of pathogen and reducing tissue damages.

The role and mechanisms of rstA in the intracellular survival of fish pathogenic Aeromonas hydrophila.

In this study, RNAi technology was used to silence the gene rstA in Aeromonas hydrophila. The strain rstA-RNAi displayed significant decrease in intracellular survival compared with that of the wild-type strain B11. Transcriptome analysis explored that the expression of some important anti-stress protein genes was significantly upregulated in rstA-RNAi compared with the wild-type strain, while the expression of the genes related to iron acquisition and type VI secretion system was significantly downregulated. Further study found that under low pH and H2 O2 stress, the anti-stress protein genes were expressed at a low level in rstA-RNAi, the growth ability of rstA-RNAi was also significantly lower than that of wild-type strain. The results also displayed that with the fluctuation of iron concentration, the expression of some genes related to iron acquisition remained at a low level in rstA-RNAi, and the growth ability of rstA-RNAi was lower than that of the wild-type strain under the same culture conditions, indicating rstA can regulate iron acquisition and further affect the bacteria growth. The adhesion ability of rstA-RNAi to fish macrophages was reduced, suggesting rstA may be also affect the formation of type VI secretion system of A. hydrophila.

flgC gene is involved in the virulence regulation of Pseudomonas plecoglossicida and affects the immune response of Epinephelus coioides.

As a pathogen of cultured teleosts, Pseudomonas plecoglossicida has caused significant economic losses. flgC plays an important role in encoding flagellar basal-body rod proteins. Our previous studies revealed the high expression of P. plecoglossicida flgC in infected Epinephelus coioides. To explore the role of flgC in the virulence of P. plecoglossicida and the immune response of E. coioides to the infection of P. plecoglossicida, flgC gene of P. plecoglossicida was knocked down by RNA interference (RNAi). The results showed that the flgC gene in all four mutants of P. plecoglossicida was significantly knocked down, and the mutant with the best knockdown efficiency of 94.3% was selected for subsequent studies. Compared with the NZBD9 strain of P. plecoglossicida, the flgC-RNAi strain showed a significantly decrease in chemotaxis, motility, adhesion, and biofilm formation. Furthermore, compared with the E. coioides infected with the NZBD9 strain, the infection of flgC-RNAi strain resulted in the infected E. coioides a 1.5-day delay in the time of first death and an 80% increase in survival rate, far fewer white nodules upon the spleen surfaces, and lower pathogen load in the spleens. RNAi of flgC significantly influenced the metabolome and transcriptome of the spleen in infected E. coioides. KEGG enrichment analysis exhibited that the Toll-like receptor signaling pathway was the most enriched immune pathway; the most significantly enriched metabolic pathways were associated with Linoleic acid metabolism, Choline metabolism in cancer, and Glycerophospholipid metabolism. Further combined analysis of transcriptome and metabolome indicated significant correlations among pantothenate and CoA biosynthesis, beta-alanine metabolism, lysosome metabolites, and related genes. These results suggested that flgC was a pathogenic gene of P. plecoglossicida; flgC was associated with the regulation of chemotaxis, motility, biofilm formation, and adhesion; flgC influenced the immune response of E. coioides to the infection of P. plecoglossicida.

Effect of the Type VI Secretion System Secreted Protein Hcp on the Virulence of Aeromonas salmonicida.

Aeromonas salmonicida, a psychrophilic bacterial pathogen, is widely distributed in marine freshwater, causing serious economic losses to major salmon farming areas in the world. At present, it is still one of the most important pathogens threatening salmon farming. Hcp (haemolysin-coregulated protein) is an effector protein in the type-VI secretion system (T6SS), which is secreted by T6SS and functions as its structural component. The results of our previous genomic sequencing showed that hcp existed in the mesophilic A. salmonicida SRW-OG1 isolated from naturally infected Epinephelus coioides. To further explore the role of Hcp in A. salmonicida SRW-OG1, we constructed an hcp-RNAi strain and verified its effect on the virulence of A. salmonicida. The results showed that compared with the wild strain, the hcp-RNAi strain suffered from different degrees of decreased adhesion, growth, biofilm formation, extracellular product secretion, and virulence. It was suggested that hcp may be an important virulence gene of A. salmonicida SRW-OG1.

α-MSH is partially involved in the immunomodulation of Nile tilapia (Oreochromis niloticus) antibacterial immunity.

α-Melanocyte-stimulating hormone (α-MSH) is a well-studied neuropeptide controlling skin and hair color. Besides, numerous immunomodulation roles of α-MSH were recorded in humans and mice. However, the regulatory effects of α-MSH in teleost immunity haven't been well elucidated. In this study, several precursor molecules of α-MSH (POMCs) and its receptors (MCRs) in Nile tilapia (Oreochromis niloticus) were characterized, and their expression characteristics and specific functions on antibacterial immunity were determined. Overall, POMCs and MCRs were principally detected in the brain, skin, and liver, and were remarkably promoted post Streptococcus agalactiae infection. However, tiny POMCs and MCRs were observed in tilapia immune organs (head kidney and spleen) or lymphocytes, and no evident immunomodulation effect was detected in vitro. Moreover, the in vivo challenge experiments revealed that α-MSH protects tilapia from bacterial infection by regulating responses in the brain and intestine. This study lays theoretical data for a deeper comprehension of the immunomodulation mechanisms of teleost α-MSH and the evolutional process of the vertebrate melanocortin system.

Transcriptomic and metabolomic insights into the role of the flgK gene in the pathogenicity of Pseudomonas plecoglossicida to orange-spotted grouper (Epinephelus coioides).

Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker (Larimichthys crocea) and orange-spotted grouper (Epinephelus coioides). Previously, RNA sequencing showed that P. plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection. To explore the role of flgK in P. plecoglossicida pathogenicity, RNA interference (RNAi) was performed to silence the P. plecoglossicida flgK gene, and the mutant (flgK-RNAi strain) with the best silencing efficiency (89.40%) was chosen for further study. Results showed that flgK gene silencing significantly attenuated P. plecoglossicida motility, adhesion, and biofilm formation. Compared to those fish infected with the wild-type strain of P. plecoglossicida, orange-spotted grouper infected with the flgK-RNAi strain showed a 55% increase in the survival rate and a one-day delay in time of first death, with fewer pathogens in the spleen and fewer white spots on the spleen surface. RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase (MAPK) signaling pathway was related to multiple immune-related pathways. Furthermore, arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways. These findings suggest that flgK is a virulence gene of P. plecoglossicida. Furthermore, flgK appears to be involved in the regulation of motility, adhesion, and biofilm formation in P. plecoglossicida, as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P. plecoglossicida infection.

Involvement and characterization of NLRCs and pyroptosis-related genes in Nile tilapia (Oreochromis niloticus) immune response.

Pyroptosis is an inflammatory and programmed cell death initiated by the formation of the inflammasome, which consists of NLR, ASC, and Caspase. Pyroptosis has received growing attention due to its association with innate immunity and various diseases. However, the involvement and induction of the NLRCs and pyroptosis-related genes in fish immunity remain poorly studied. In this study, several NLRCs and pyroptosis-related genes in Nile tilapia (Oreochromis niloticus) were identified and characterized. Their involvement in bacterial infection and expression profiles in Nile tilapia lymphocyte responses were also assessed. Overall, three NLRC members (NOD1, NOD2, and NLRC3) and five pyroptosis-related genes (ASC1, Caspase1, Gsdme, NLRP3, and NLRP14) in Nile tilapia were cloned and characterized. The transcript levels of these molecules were broadly distributed in various tissues with comparatively high expression in the gills, intestine, and spleen. Their transcripts were also induced during Streptococcus agalactiae or Aeromonas hydrophila infection. Moreover, they were primarily expressed in T cells, NCCs, and Mo/Mφ and showed antibacterial and partially antiviral responses. The present study lays a theoretical foundation for further investigation of the pyroptosis mechanisms in fish as well as the evolution of the antiviral roles of pyroptosis in vertebrates.

Metabolomics insights into the interaction between Pseudomonas plecoglossicida and Epinephelus coioides.

As a highly infectious epidemic in aquaculture, Pseudomonas plecoglossicida infection results in high mortality of teleosts and serious economic losses. Host-pathogen interactions shape the outcome of an infection, yet we still understand little about the molecular mechanism of these pathogen-mediated processes. Here, a P. plecoglossicida strain (NZBD9) and Epinephelus coioides were investigated as a model system to characterize pathogen-induced host metabolic remodeling over the course of infection. We present a non-targeted metabolomics profiling of E. coioides spleens from uninfected E. coioides and those infected with wild-type and clpV-RNA interference (RNAi) strains. The most significant changes of E. coioides upon infection were associated with amino acids, lysophospatidylcholines, and unsaturated fatty acids, involving disturbances in host nutritional utilization and immune responses. Dihydrosphingosine and fatty acid 16:2 were screened as potential biomarkers for assessing P. plecoglossicida infection. The silencing of the P. plecoglossicida clpV gene significantly recovered the lipid metabolism of infected E. coioides. This comprehensive metabolomics study provides novel insights into how P. plecoglossicida shape host metabolism to support their survival and replication and highlights the potential of the virulence gene clpV in the treatment of P. plecoglossicida infection in aquaculture.

Function of the rpoD gene in Pseudomonas plecoglossicida pathogenicity and Epinephelus coioides immune response.

Pseudomonas plecoglossicida is a Gram-negative pathogenic bacterium that causes visceral white spot disease in several marine fish species, resulting in high mortality and financial loss. Based on previous RNA sequencing (RNA-seq) results, rpoD gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that rpoD may contribute to bacterial pathogenicity. To investigate the role of this gene, five specific short hairpin RNAs (shRNAs) were designed and synthesized based on the rpoD gene sequence, with all five mutants exhibiting a significant decrease in rpoD gene expression in P. plecoglossicida. The mutant with the highest silencing efficiency (89.2%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain NZBD9, silencing rpoD in the rpoD-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of rpoD also resulted in a 25% increase in the survival rate, a one-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface of infected orange-spotted groupers (Epinephelus coioides). In addition, rpoD expression and pathogen load were significantly lower in the spleens of E. coioides infected with the rpoD-RNAi strain than with the WT strain of P. plecoglossicida. We performed RNA-seq of E. coioides spleens infected with different P. plecoglossicida strains. Results showed that rpoD silencing in P. plecoglossicida led to a significant change in the infected spleen transcriptomes. In addition, comparative transcriptome analysis showed that silencing rpoD caused significant changes in complement and coagulation cascades and the IL-17 signaling pathway. Thus, this study revealed the effects of the rpoD gene on P. plecoglossicida pathogenicity and identified the main pathway involved in the immune response of E. coioides.

The role and mechanisms of the two-component system EnvZ/OmpR on the intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila infections are common in aquaculture. Our previous studies found that the A. hydrophila B11 strain can survive in fish macrophages for at least 24 h and the two-component system EnvZ/OmpR may be involved in intracellular survival. To reveal the role and mechanism of the two-component system EnvZ/OmpR in intracellular survival of A. hydrophila, the genes of envZ/ompR were silenced by shRNAi. The results showed that the survival rates of the envZ-RNAi and ompR-RNAi strains were only 2.05% and 3.75%, respectively, which were decreased by 91% and 83.6% compared with that of the wild-type strain. The escape ability of envZ-RNAi and ompR-RNAi was also decreased by 51.4% and 19.7%, respectively. The comparative transcriptome analysis revealed that the functional genes directly related to bacterial intracellular survival mainly included the genes related to anti-stress capacity, and the genes related to Zn2+ and Mg2+ transport. Further research confirmed that two-component system EnvZ/OmpR can regulate the expression of the important molecular chaperones, such as groEL, htpG, dnaK, clpB and grpE. The expression of these molecular chaperones in wild-type strain was up-regulated with the increase in H2 O2 concentrations, while the expression of these molecular chaperones in silent strains did not change significantly. Cells that phagocytosed wild-type strain had higher ROS content than cells that phagocytosed silent strains. Two-component system EnvZ/OmpR could also regulate zinc transporter (znuA, znuB, znuC) and zinc efflux protein (zntA) to maintain zinc homeostasis in cells, thus affecting the ability of bacteria to survive in phagocytes. Moreover, two-component system EnvZ/OmpR could affect the growth and intracellular survival of A. hydrophila by regulating the expression of MgtA, MgtC and MgtE and participating in bacterial Mg2+ homeostasis in fish macrophages.

Effect of Ferredoxin Receptor FusA on the Virulence Mechanism of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is an aerobic Gram-negative bacterium, which is the pathogen of "Visceral white spot disease" in large yellow croaker. P. plecoglossicida is a temperature-dependent bacterial pathogen in fish, which not only reduces the yield of large yellow croaker but also causes continuous transmission of the disease, seriously endangering the healthy development of fisheries. In this study, a mutant strain of fusA was constructed using homologous recombination technology. The results showed that knockout of P. plecoglossicida fusA significantly affected the ability of growth, adhesion, and biofilm formation. Temperature, pH, H2O2, heavy metals, and the iron-chelating agent were used to treat the wild type of P. plecoglossicida; the results showed that the expression of fusA was significantly reduced at 4°C, 12°C, and 37°C. The expression of fusA was significantly increased at pH 4 and 5. Cu2+ has a significant inducing effect on the expression of fusA, but Pb2+ has no obvious effect; the expression of fusA was significantly upregulated under different concentrations of H2O2. The expression of the fusA gene was significantly upregulated in the 0.5~4-µmol/l iron-chelating agent. The expression level of the fusA gene was significantly upregulated after the logarithmic phase. It was suggested that fusA included in the TBDR family not only was involved in the transport of ferredoxin but also played important roles in the pathogenicity and environment adaptation of P. plecoglossicida.

Immune responses and inorganic ion transport regulations of Epinephelus coioides in response to L321_RS13075 gene of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a well-known pathogen of viscera granulomas disease in fish, which has led to severe economic losses. In our previous study, L321_RS13075 was predicted to be a key virulence gene of P. plecoglossicida during the host-pathogen interaction with Epinephelus coioides. To investigate the role of L321_RS13075 in the regulation of virulence in P. plecoglossicida, a L321_RS13075 knock-down strain was constructed. And a significant reduction in the ability of colonization, intracellular survival, motility, biofilm formation, and adhesion was detected in the L321_RS13075 knock-down strain. Compared with the wild-type strain, the silence of L321_RS13075 in P. plecoglossicida resulted in a significant change in the transcriptome of infected Epinephelus coioides (E. coioides). Results of COG and GO analysis on E. coioides showed that genes related to immune responses and inorganic ion transport were significantly affected by L321_RS13075 of P. plecoglossicida. Meanwhile, the interactions of the genes related to immune responses and inorganic ion transport were predicted, and the important hub genes were identified. Taken together, the results indicated that L321_RS13075 was a virulent gene of P. plecoglossicida, which significantly affected the immune responses and inorganic ion transport in E. coioides.

The contribution of exbB gene to pathogenicity of Pseudomonas plecoglossicida and its interactions with Epinephelus coioides.

To study the roles of the exbB gene in Pseudomonas plecoglossicida during interactions with Epinephelus coioides, five short hairpin RNAs (shRNAs) were designed and synthesized to silence the exbB gene in P. plecoglossicida which resulted in significant reductions in exbB mRNA expression. The mutant with the best silencing efficiency (89.3%) was selected for further study. Silencing exbB in the exbB-RNA interference (RNAi) strain resulted in a 70% increase in the survival rate and a 3-day delay in the onset of infection in E. coioides. Silencing of the exbB gene also resulted in a significant decrease in the number of white spots on the spleen surface and in the spleen pathogen load. The results of dual RNA-seq showed that exbB silencing in P. plecoglossicida also resulted in a significant change in both the pathogen and host transcriptomes in the spleens of infected E. coioides. Comparative transcriptome analysis showed that silencing exbB caused significant changes in multiple signaling molecules and interaction- and immune system-related genes in E. coioides. Gene silencing also resulted in the differential expression of flagellar assembly and the bacterial secretion system in P. plecoglossicida during the infection period, and most of the DEGs were down-regulation. These host-pathogen interactions may make it easier for E. coioides to eliminate the exbB-RNAi strain of P. plecoglossicida, suggesting a significant decrease in the pathogenicity of this strain. These results indicated that exbB was a virulence gene of P. plecoglossicida which contributed a lot in the pathogen-host interactions with E. coioides.

Full-Length Transcriptome: A Reliable Alternative for Single-Cell RNA-Seq Analysis in the Spleen of Teleost Without Reference Genome.

Fish is considered as a supreme model for clarifying the evolution and regulatory mechanism of vertebrate immunity. However, the knowledge of distinct immune cell populations in fish is still limited, and further development of techniques advancing the identification of fish immune cell populations and their functions are required. Single cell RNA-seq (scRNA-seq) has provided a new approach for effective in-depth identification and characterization of cell subpopulations. Current approaches for scRNA-seq data analysis usually rely on comparison with a reference genome and hence are not suited for samples without any reference genome, which is currently very common in fish research. Here, we present an alternative, i.e. scRNA-seq data analysis with a full-length transcriptome as a reference, and evaluate this approach on samples from Epinephelus coioides-a teleost without any published genome. We show that it reconstructs well most of the present transcripts in the scRNA-seq data achieving a sensitivity equivalent to approaches relying on genome alignments of related species. Based on cell heterogeneity and known markers, we characterized four cell types: T cells, B cells, monocytes/macrophages (Mo/MΦ) and NCC (non-specific cytotoxic cells). Further analysis indicated the presence of two subsets of Mo/MΦ including M1 and M2 type, as well as four subsets in B cells, i.e. mature B cells, immature B cells, pre B cells and early-pre B cells. Our research will provide new clues for understanding biological characteristics, development and function of immune cell populations of teleost. Furthermore, our approach provides a reliable alternative for scRNA-seq data analysis in teleost for which no reference genome is currently available.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides.

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

The Effect of tonB Gene on the Virulence of Pseudomonas plecoglossicida and the Immune Response of Epinephelus coioides.

Pseudomonas plecoglossicida is the causative agent of "visceral white spot disease" in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.

Integration of RNA-seq and RNAi reveals the contribution of znuA gene to the pathogenicity of Pseudomonas plecoglossicida and to the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 105  cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.