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.

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.

The hybrid offspring of barbel chub Squaliobarbus curriculus and grass carp Ctenopharyngodon idella exhibit stronger resistance to the grass carp reovirus (GCRV) infection than grass carp. Toll-like receptors (TLRs) play indispensable roles in the antiviral immunity of fish. In this study, the structures and antiviral immune functions of barbel chub TLR19 (ScTLR19) and grass carp TLR19 (CiTLR19) were compared. The amino acid sequence of ScTLR19 shared high similarity (97.4%) and identity (94.0%) with that of CiTLR19, and a phylogenetic tree revealed the close evolutionary relationship between ScTLR19 and CiTLR19. Protein domain composition analyses showed that ScTLR19 possessed an additional leucine-rich repeat (designated as LRR9) located at amino acid positions 654–677 in the extracellular region, which was absent in CiTLR19. Multiple sequence alignment and three-dimensional structure comparison also indicated that the extracellular regions of ScTLR19 and CiTLR19 exhibited greater differences compared to their intracellular regions. Molecular docking revealed that the extracellular region of ScTLR19 (docking score = −512.31) showed a stronger tendency for binding with polyI:C, compared to the extracellular region of CiTLR19 (docking score = −474.90). Replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 reduced the binding activity of ScTLR19 to polyI:C, as confirmed by an ELISA. Moreover, overexpression experiments suggested that ScTLR19 could regulate both the IRF3–TRIF and IRF3–MyD88 signaling pathways during GCRV infection, while CiTLR19 only regulated the IRF3–MyD88 signaling pathway. Importantly, replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 altered the expression regulation on IRF3, MyD88, and TRIF during GCRV infection. These findings collectively reveal the structural and functional differences between ScTLR19 and CiTLR19, and they may provide data to support a deeper understanding of the molecular mechanisms underlying the differences in GCRV resistance between barbel chub and grass carp, as well as the genetic basis for the heterosis of GCRV resistance in their hybrid offspring.

Grass carp (Ctenopharyngodon idella) aquaculture faces significant challenges due to grass carp hemorrhagic disease, which is instigated by the grass carp reovirus (GCRV). A major contributor to the high mortality rates associated with this disease is an excessive inflammatory response. Autophagy contributes to host disease resistance partly through modulating inflammation, in which autophagy-related gene 5 (ATG5) serves as a core component. In this study, we generated an ATG5-edited cell model with biallelic frameshift mutations in C. idella kidney (CIK) cells using the CRISPR/Cas9 system. Transcriptome and qPCR analyses indicated that ATG5 gene editing significantly influenced multiple biological pathways, including oxidative phosphorylation, phagosome formation, and arachidonic acid metabolism, and altered the expression of inflammation-related genes such as IL-1β, IL-8, TNF-α, IL-10, and TGF-β in CIK cells during GCRV infection. Further investigations through Western blot and ubiquitination assays demonstrated that ATG5 gene editing led to a notable decrease in the LC3-II/LC3-I ratio in CIK cells, along with a buildup of pro-IL-1β protein. The overexpression of grass carp ATG5 and IL-1β in HEK-293T cells reduced the ubiquitination of grass carp pro-IL-1β, facilitating its accumulation. Collectively, our results indicate that grass carp ATG5 modulates the expression of multiple inflammatory mediators and may shape pro-IL-1β protein abundance in a ubiquitination-associated manner. This study provides insights into autophagy-linked inflammatory regulation underlying grass carp hemorrhagic disease.

Neolissochilus heterostomus, a cyprinid fish endemic to Yunnan Province, China, is highly valued for both ornamental and edible purposes, yet its wild populations are currently declining and classified as endangered. In this study, we present a chromosome-level genome assembly of N. heterostomus, utilizing the PacBio HiFi and Hi-C strategies. The assembled genome spans 1793.99 Mb and is anchored to 50 chromosomes, comprising a total of 50,203 genes. Genomic features and chromosomal karyotype data recorded in the present study are consistent with an allotetraploid origin for N. heterostomus. Comparative genomics shows that N. heterostomus shares a close phylogenetic relationship with another allotetraploid fish, the common carp Cyprinus carpio. In total, 4687 expanded and 1365 contracted gene families were identified in N. heterostomus. Further enrichment analyses indicated an overrepresentation of metabolism-related pathways among the expanded and positively selected gene families, whereas the contracted gene families were enriched in reproduction- and embryonic development-related pathways. Reproductive observations further indicate that N. heterostomus produces fewer eggs than other cyprinid species characterized by relatively long embryonic development periods (106.1 h; accumulated temperature: 2260.6 °C·h), including common carp, goldfish (Carassius auratus), grass carp (Ctenopharyngodon idella), pond loach (Misgurnus anguillicaudatus), and blunt snout bream (Megalobrama amblycephala). These findings suggest a potential association between metabolism-related genomic features and reproductive traits, although functional validation remains necessary. Overall, this study may offer insights into the polyploidization, metabolic, and reproductive traits of N. heterostomus, thereby providing genomic and biological resources that may support future studies relevant to the conservation and management of this endangered species.