The Teleost CXCL13-CXCR5 Axis Induces Inflammatory Cytokine Expression through the Akt-NF-κB, p38-AP-1, and p38-NF-κB Pathways.

The ancestors of chemokines originate in the most primitive of vertebrates, which has recently attracted great interest in the immune functions and the underlying mechanisms of fish chemokines. In the current study, we identified an evolutionarily conserved chemokine, CiCXCL13, from a teleost fish, grass carp. CiCXCL13 was characterized by a typical SCY (small cytokine CXC) domain and four cysteine residues (C34, C36, C61, C77), with the first two cysteines separated by a random amino acid residue, although it shared 24.2-54.8% identity with the counterparts from other vertebrates. CiCXCL13 was an inducible chemokine, whose expression was significantly upregulated in the immune tissues of grass carps after grass carp reovirus infection. CiCXCL13 could bind to the membrane of grass carp head kidney leukocytes and promote cell migration, NO release, and the expression of >15 inflammatory cytokines, including IL-1ß, TNF-α, IL-10 and TGF-ß1, thus regulating the inflammatory response. Mechanistically, CiCXCL13 interacted with its evolutionarily conserved receptor CiCXCR5 and activated the Akt-NF-κB and p38-AP-1 pathways, as well as a previously unrevealed p38-NF-κB pathway, to efficiently induce inflammatory cytokine expression, which was distinct from that reported in mammals. Zebrafish CXCL13 induced inflammatory cytokine expression through Akt, p38, NF-κB, and AP-1 as CiCXCL13. Meanwhile, the CiCXCL13-CiCXCR5 axis-mediated inflammatory activity was negatively shaped by grass carp atypical chemokine receptor 2 (CiACKR2). The present study is, to our knowledge, the first to comprehensively define the immune function of CXCL13 in inflammatory regulation and the underlying mechanism in teleosts, and it provides a valuable perspective on the evolution and biology of fish chemokines.

An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases.

Correction: An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

[This corrects the article DOI: 10.1371/journal.ppat.1011320.].

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection.

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Genome-wide identification of Gα family in grass carp (Ctenopharyngodon idella) and reproductive regulation functional characteristics of Cignaq.

BACKGROUND: The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp. RESULTS: Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression. CONCLUSIONS: These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts.

The role of MASP1 in the complement system and expression characteristics in response to GCRV infection in grass carp.

The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30-91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation.

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.

Identification of the C1qDC gene family in grass carp (Ctenopharyngodon idellus) and the response of C1qA, C1qB, and C1qC to GCRV infection in vivo and in vitro.

Proteins from the C1q domain-containing (C1qDC) family recognize self-, non-self-, and altered-self ligands and serves as an initiator molecule for the classical complement pathway as well as recognizing immune complexes. In this study, C1qDC gene family members were identified and analyzed in grass carp (Ctenopharyngodon idellus). Members of the C1q subfamily were cloned, and their response to infection with the grass carp virus was investigated. In the grass carp genome, 54 C1qDC genes and 67 isoforms have been identified. Most were located on chromosome 3, with 52 shared zebrafish homologies. Seven substantially differentially expressed C1qDC family genes were identified in the transcriptomes of cytokine-induced killer (CIK) cells infected with grass carp reovirus (GCRV), all of which exhibited sustained upregulation. The opening reading frames of grass carp C1qA, C1qB, and C1qC, belonging to the C1q subfamily, were determined to be 738, 732, and 735 base pairs, encoding 245, 243, and 244 amino acids with molecular weights of 25.81 kDa, 25.63 kDa and 26.16 kDa, respectively. Three genes were detected in the nine collected tissues, and their expression patterns were similar, with the highest expression levels observed in the spleen. In vivo after GCRV infection showed expression trends of C1qA, C1qB, and C1qC in the liver, spleen, and kidney. An N-type pattern in the liver and kidney was characterized by an initial increase followed by a decrease, with the highest expression occurring during the recovering period, and a V-type pattern in the spleen with the lowest expression levels during the death period. In vitro, after GCRV infection showed expression trends of C1qA, C1qB, and C1qC, and this gradually increased within the first 24 h, with a notable increase observed at the 24 h time point. After CIK cells incubation with purified recombinant proteins, rC1qA, rC1qB, and rC1qC for 3 h, followed by GCRV inoculation, the GCRV replication indicated that rC1qC exerted a substantial inhibitory effect on viral replication in CIK cells after 24 h of GCRV inoculation. These findings offer valuable insights into the structure, evolution, and function of the C1qDC family genes and provide a foundational understanding of the immune function of C1q in grass carp.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Structural comparison and expression function analysis of BF/C2 in Ctenopharyngodon idella and Squaliobarbus curriculus.

Ctenopharyngodon idella and Squaliobarbus curriculus, members of the Cyprinidae family and Yaroideae subfamily, have shown different levels of resistance to grass carp reo virus (GCRV), with S. curriculus exhibiting higher resilience. In the pursuit to explore the distinctions in the structural and expression traits of BF/C2 (A,B) between the two species, we conducted an analysis involving the cloning and examination of various coding sequences (CDS). We successfully cloned the CDS of ci-BF/C2A and ci-BF/C2B from C. idella, which spanned 2259 bp and 2514 bp respectively, encoding 752 and 837 amino acids. Similarly, the CDS of sc-BF/C2A and sc-BF/C2B from S. curriculus were cloned, featuring lengths of 1353 bp and 2517 bp and encoding 450 and 838 amino acids, respectively. A chromosome collinearity assessment revealed that ci-BF/C2A demonstrated collinearity with sc-BF/C2A, a finding not replicated with ci-BF/C2B and sc-BF/C2B. Delving into gene structure, we discerned that ci-BF/C2A harbored a greater number of Tryp_SPc domains compared to sc-BF/C2A. Following this, we engineered and purified six prokaryotic recombinant proteins: CI-BF/C2A, CI-BF/C2A1 (a variant resulting from the deletion of the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2A2 (representing the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2B, SC-BF/C2A, and SC-BF/C2B. Through serum co-incubation tests with these recombinant proteins, we established the activation of the complement marker C3 in each case. Utilizing fluorescence quantitative expression analysis, we observed ubiquitous expression of ci-BF/C2A and ci-BF/C2B across all grass carp tissues, predominantly in the liver. This pattern mirrored in S. curriculus, where sc-BF/C2A was highly expressed in the gills, and sc-BF/C2B manifested notably in the liver. Kidney cell infection experiments on both species revealed enhanced resistance to GCRV post-incubation with the recombinant proteins. Notably, cells treated with SC-BF/C2 (A, B) exhibited pronounced resilience compared to those treated with CI-BF/C2 (A, B, A1, A2). However, cells incubated with CI-BF/C2A1 and CI-BF/C2A2 showed strengthen resistance relative to cells treated with CI-BF/C2A and CI-BF/C2B. In GCRV infection trials on grass carp, ci-BF/C2A and ci-BF/C2B expressions reached a zenith on the seventh day post-infection, highlighting a distinctive functional mode in immune defense against GCRV infection orchestrated by BF/C2. The empirical data underscores the pivotal role of the Tryp_SPc domain in immune responses to GCRV infection, pinpointing its influence on ci-BF/C2A expression. Conclusively, this investigation provides a foundational understanding of the unique immune function characteristics of BF/C2 in grass carp, paving the way for further scholarly exploration in this realm.

Nucleophosmin 1a translocated from nucleus to cytoplasm and facilitate GCRV replication.

To decipher the functional characterization of Nucleophosmin 1a (NPM1a) from grass carp (Ctenopharyngodon idellus) (CiNPM1a), its cDNA was cloned and bioinformatic analysis were conducted. The full-length cDNA sequence of CiNPM1a is 1732 bp, which encodes 307 amino acids. CiNPM1a contains conserved domains of Nucleoplasmin domain, NPM1-C terminal domain, as well as nuclear localization signals, nuclear export signal (NES) and acid patches. There are 52 and 20 consensus amino acids exist in the Nucleoplasmin domain and the NPM1-C terminal domain of all blasted species. In addition, the immune function of CiNPM1a were analyzed. The Ciirf7, Ciifn1 and Ciifn2 transcription was inhibited, whereas the vp2 and vp7 expressions were enhanced in CiNPM1a overexpressing cells after GCRV infection (P < 0.05). Moreover, the Ciirf7, Ciifn1 and Ciifn2 mRNA levels were significantly up-regulated, but the vp2 and vp7 expressions were significantly down-regulated in CiNPM1a knockdown cells after infection. This indicated that CiNPM1a played negative roles in the induction of Type I IFN reaction and thus the GCRV replication. Finally, the NES domain that affect the nucleous-cytoplasm shuttle and the replication of GCRV were investigated. The deletion of NES1 and NES(1 + 2+3) absolutely limited the transloacation of CiNPM1a△NES1 protein and CiNPM1a △NES(1 + 2+3) protein to cytoplasm after infection, and the deletion of NES2 resulted in partially limitation of protein shuttle. In general, Ciirf3, Ciirf7, Ciifn1 and Ciifn2 expressions were enhanced in the CiNPM1a△NES1, CiNPM1a△NES2 and CiNPM1a△NES3 overexpression groups, and the deletion of functional domains in CiNPM1a led to significantly reduction of the vp2 and vp7 replication. The results indicated that CiNPM1a may be a target molecular for GCRV infection curation, and a candidate molecular for resistance strain breeding of grass carp.

Effect of dietary replacement of fish meal by poultry by-product meal on the growth performance, immunity, and intestinal health of juvenile red swamp crayfish, procambarus clarkia.

The present study was conducted to investigate the dietary replacement of fish meal with poultry by-product meal (PBM) on the growth performance, immunity, antioxidant properties, and intestinal health of red swamp crayfish (Procambarus clarkia). A diet containing 20% fish meal (FM) and complex plant ingredients as the main protein resources was set as the FM group (crude protein 32%, crude lipid 6%). Four diets replacing 25%, 50%, 75%, and 100% fish meal of the FM diet with PBM were set as the PBM25, PBM50, PBM75, and PBM100 groups, respectively. Compared to the FM group, the PBM100 diet significantly decreased growth performance and feed utilization of crayfish, while markedly increasing the activity of serum aspartate aminotransferase. The immune response was depressed in crayfish fed the PBM100 diet as the activities of serum lysozyme and phenoloxidase, gene expression of anti-lipopolysaccharide factors (alf), cyclophilin A (cypa), crustin, and hemocyanin-1 (hep-1) in hepatopancreas were remarkably decreased. The activities of antioxidases and expression of antioxidant-relevant genes in the hepatopancreas were not influenced by PBM inclusion. Crayfish fed different diets exhibited no obvious symptoms of enteritis, but the PBM100 diet destructed intestinal morphology by significantly decreasing the average length of longitudinal ridges. The α-diversity and overall community structure were not significantly influenced but variations were found in the relative abundance of some genera by PBM inclusion. In summary, CAP could successfully replace 75% dietary FM in a basal diet containing 20% fish meal, while higher CAP level compromised growth performance, immunity, and intestinal histology of crayfish.

Functional and expression analysis reveals the involvement of integrin αvß3 in antiviral immunity of grass carp (Ctenopharyngodon idella).

Integrins are α-ß heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvß3 (GCαvß3) originally assembled by αv (GCαv) and ß3 (GCß3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCß3 shared high similarity (75.2-95.1%) and identity (58.6-90.7%) with their homologs from other vertebrates. Both GCαv and GCß3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCß3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCß3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvß3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvß3 did. In addition, both GCαv and GCß3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvß3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.

Sequence, Expression, and Anti-GCRV Function of the Ferritin from the Grass Carp, Ctenopharyngodon idellus.

Ferritin possesses an immune function to defend against pathogen infection. To elucidate the immunity-protecting roles of ferritin from Ctenopharyngodon idellus (Ciferritin) against virus infection, the cDNA and promoter sequences of Ciferritin were determined, and the correlations between Ciferrtin expressions and promoter methylation levels were analyzed. In addition, the functional role of Ciferrtin on GCRV (grass carp reovirus) infection was assessed. The full-length cDNA of Ciferritin is 1053 bp, consists of a 531 bp open-reading frame, and encodes 176 amino acids. Ciferritin showed the highest sequence identity with the ferritin middle subunit of Mylopharyngodon piceus (93.56%), followed by the subunits of Megalobrama amblycephala and Sinocyclocheilus rhinocerous. Ciferritin contains a conserved ferritin domain (interval: 10−94 aa), and the caspase recruitment domain (CARD) and Rubrerythrin domain were also predicted. In the spleen and kidney, significantly higher Ciferritin expressions were observed at 6, 12, 24, or 168 h post GCRV infection than those in the PBS injection group (p < 0.05). The Ciferrtin expression level in the progeny of maternal-immunized grass carp was significantly higher than that in the progeny of common grass carp (p < 0.05). Ciferritin promoter methylation level in the progeny from common grass carp was 1.27 ± 0.15, and in the progeny of the maternal-immunized group was 1.00 ± 0.14. In addition, methylation levels of "CpG9" and "CpG10" loci were significantly lower in the progeny of maternal-immunized fish than those in the common group. Except for the "CpG5", methylation levels of all other detected "CpG" loci negatively correlated with Ciferritin expression levels. Furthermore, the total methylation level of "CpG1−10" negatively correlated with the Ciferritin expressions. The Ciferritin expression level was significantly up-regulated, and the VP7 protein levels were significantly reduced, at 24 h post GCRV infection in the Ciferritin over-expression cells (p < 0.05). The results from the present study provide sequence, epigenetic modification and expression, and anti-GCRV functional information of Ciferritin, which provide a basis for achieving resistance to GCRV in grass carp breeding.

Functional and Expressional Analyses Reveal the Distinct Role of Complement Factor I in Regulating Complement System Activation during GCRV Infection in Ctenopharyngodon idella.

Complement factor I (CFI), a complement inhibitor, is well known for regulating the complement system activation by degrading complement component 3b (C3b) in animal serum, thus becoming involved in innate defense. Nevertheless, the functional mechanisms of CFI in the complement system and in host-pathogen interactions are far from being clarified in teleost fish. In the present study, we cloned and characterized the CFI gene, CiCFI, from grass carp (Ctenopharyngodon idella) and analyzed its function in degrading serum C3b and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiCFI was found to be 2121 bp, encoding 706 amino acids with a molecular mass of 79.06 kDa. The pairwise alignments showed that CiCFI shared the highest identity (66.9%) with CFI from Carassius gibelio and the highest similarity (78.7%) with CFI from Danio rerio. The CiCFI protein was characterized by a conserved functional core Tryp_SPc domain with the catalytic triad and substrate binding sites. Phylogenetic analysis indicated that CiCFI and the homologs CFIs from other teleost fish formed a distinct evolutionary branch. Similar with the CFIs reported in mammals, the recombinant CiCFI protein could significantly reduce the C3b content in the serum, demonstrating the conserved function of CiCFI in the complement system in the grass carp. CiCFI mRNA and protein showed the highest expression level in the liver. After GCRV infection, the mRNA expressions of CiCFI were first down-regulated, then up-regulated, and then down-regulated to the initial level, while the protein expression levels maintained an overall downward trend to the late stage of infection in the liver of grass carps. Unexpectedly, the protein levels of CiCFI were also continuously down-regulated in the serum of grass carps during GCRV infection, while the content of serum C3b proteins first increases and then returns to the initial level, suggesting a distinct role of CiCFI in regulating complement activation and fish-virus interaction. Combining our previous results that complement factor D, a complement enhancer, shows continuously up-regulated expression levels in grass carps during GCRV infection, and this study may provide the further essential data for the full picture of complex complement regulation mechanism mediated by Df and CFI of the grass carp during pathogen infection.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

Functional Identification of Complement Factor D and Analysis of Its Expression during GCRV Infection in Grass Carp (Ctenopharyngodon idella).

Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, CiDf, from grass carp (Ctenopharyngodon idella) and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiDf was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. CiDf harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that CiDf shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami. Phylogenetic analysis indicated that CiDf and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the CiDf gene structure is characterized by four introns and five exons. The incubation of recombinant CiDf protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of CiDf in the AP in promoting C3 cleavage, similar to Dfs in mammals. CiDf mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of CiDf mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of CiDf and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp C. idella.

The microRNA-200 cluster on chromosome 23 is required for oocyte maturation and ovulation in zebrafish†.

The reproductive process is usually controlled by the hypothalamic-pituitary-gonad axis in vertebrates, while Kiss/gonadotropin-releasing hormone (GnRH) system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of follicle stimulating hormone/luteinizing hormone (LH) expression in fish species is still unknown. Here, we identified miR-200s on chromosome 23 (chr23-miR-200s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200s (chr23-miR-200s-KO) resulted in dysregulated expression of luteinizing hormone beta lhb (luteinizing hormone beta) and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of human chorionic gonadotropin (hCG) could rescue the defects of oocyte maturation in chr23-miR-200s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction, or co-injection of hCG with synthetic oxytocin and vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200s in controlling oocyte maturation by regulation LH expression, and oxytocin and vasotocin are potentially responsible for the ovulation in fish species.

Sequence and expression analysis of the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 from the barbel chub Squaliobarbus curriculus.

MDA5 is a cytoplasmic viral double-stranded RNA recognition receptor that plays a pivotal role in the aquatic animal innate immune system. To decipher the role of MDA5 of Squaliobarbus curriculus (ScMDA5) in the immune response, full-length cDNA of ScMDA5 was cloned using the RACE technology, mRNA and protein expression levels of ScMDA5 signalling pathway members in response to stimulation were detected and effects of overexpression of ScMDA5 on the immune response were investigated. ScMDA5 comprises 3597 bp and is composed of an open reading frame (2958 nucleotides long) that translates into a putative peptide of 985 amino acid residues. ScMDA5 possesses two N-terminal caspase-recruiting domains, DEAD-like helicases superfamily, helicase superfamily C-terminal and RIG-I_C-RD domains, and differences in these domains among species were mainly observed with respect to their length and location. ScMDA5 was closely clustered with those of Carassius auratus, Ctenopharyngodon idellus and Mylopharyngodon piceus. ScMDA5 transcripts were most abundant in the spleen and the lowest in the liver. Expression levels of ScMDA5 in healthy tissues were significantly correlated with those of ScIRF3, ScIRF7 and ScIFN. Besides, mRNA expression levels of ScIRF3 were significantly correlated with those of ScIRF7 (0.956, P < 0.01). Expression level changes, including downregulation, upregulation and initial upregulation followed by downregulation, were found in ScMDA5 signalling pathway molecules in tissues after grass carp reovirus infection. Protein levels of ScMDA5 were the highest in the liver and the lowest in the spleen in detected healthy tissues. Overexpression of ScMDA5 led to significantly enhanced CiIRF7 and CiMx transcription in grass carp ovary cells (P < 0.05). The results of this study helped to clarify the role of ScMDA5 in the immune reaction against grass carp reovirus and provided fundamental information for fish breeding to achieve strong resistance to infection.