In the host immune system, perforin is a cytotoxic effector molecule that eliminate virus-infected and malignant cells. Moreover, some recent studies also imply the involvement of perforin in antibacterial immunity. Common carp (Cyprinus carpio L.), one of the most economically important fish species in China, has a high susceptibility to viruses and bacteria. Thus far, in common carp, no data are available regarding the identification and immunologic function of the perforin. In the present study, the cDNA and genomic DNA sequences of three perforin isoform genes were cloned and characterized in common carp, named CcPRF1, CcPRF2 and CcPRF3. Amino acid sequences of the three CcPRFs were quite different, with identities ranged from 37.3 to 39.5%. Phylogenetic analysis showed that three CcPRFs, each in a separate sub-branch, possessed closer evolutionary relationship with other teleost perforins, especially with cyprinid fishes, than higher vertebrates. Expression analysis revealed that each CcPRF gene was differentially expressed in all of the nine tested tissues. During larvae ontogeny, each CcPRF displayed a distinct expression pattern, while with a common expression peak at 22 days post hatching (dph). Moreover, in vivo or in vitro, after stimulation with polyI:C, LPS and Aeromonas hydrophila, each CcPRF was induced significantly, with differential expression dynamics. Our findings suggest that perforin might play significant roles in larval immune system and in the immune defense of common carp against viral and bacterial pathogens. Meantime, the differential expression dynamics seem to imply possible different cellular locations or functional differences across various CcPRF isoforms.

Immunoglobulin M (IgM) is the primary antibody in fish, which is transported from epithelial cells into the external secretion system by polymeric immunoglobulin receptor (pIgR). In this study, the full length of IgM and pIgR complementary DNA sequences from leopard coral grouper (Plectropomus leopardus) was characterized, and their expression levels under Vibrio harveyi infection were studied. cDNA sequence analysis showed that the pl-IgM heavy chain cDNA contained an open reading frame (ORF) of 1797bp encoding a polypeptide of 597 amino acids consisting of a signal peptide, a variable region, and 4 constant regions. pl-pIgR (1041bp) encodes 346 amino acids with 2 Ig-like domains (ILDs). The expression levels of pl-IgM and pl-pIgR in healthy fish were mainly in the intestine and spleen of fish, which indicates the relationship between the mucosal-associated lymphoid tissue and system lymphoid tissue. Messenger RNA (mRNA) expression profiles of pl-IgM and pl-pIgR were significantly increased in the intestine, head kidney, spleen, and liver after V. harveyi infection. The cDNA sequences of two ILDs were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant pl-pIgR protein had antibacterial function against V. harveyi and Vibrio alginolyticus at 200ng/μl. Our results indicate that pl-IgM and pl-pIgR are involved in the mucosal-associated immune and systemic immune response in the antibacterial immunity of leopard coral grouper.

The leopard coral grouper (Plectropomus leopardus), an economically important marine fish, is widely farmed in Hainan Province, China. However, intensive farming has led to frequent disease outbreaks, including viral nervous necrosis, vibriosis, and enteritis, with skin ulceration a widespread and significant concern. To identify the pathogen causing skin ulceration, we collected diseased fish from Hainan and conducted histopathological and ultrastructural analyses, parasite examination, viral metagenomic sequencing, and bacterial isolation and identification. The histopathological analysis and ultrastructural observation showed that the diseased P. leopardus displayed marked pathological alterations, with significant hepatic degeneration and necrosis and splenic structural disorganization. Parasites and viruses were both excluded from being the primary pathogen. A total of 79 bacterial strains were isolated from diseased P. leopardus liver tissue, including Vibrio harveyi (38 strains, 48.1%), Vibrio owensii (17 strains, 21.5%), Vibrio rotiferianus (16 strains, 20.3%), and Photobacterium damselae subsp. damselae (8 strains, 10.1%). Infection experiments suggested that all four bacterial species could induce skin ulceration. This study demonstrates that skin ulceration in P. leopardus is associated with multiple bacterial pathogens, refining the previous view that attributed the disease solely to V. harveyi. These findings provide a foundation for developing vaccines and disease-resistant breeding strategies.

This study investigates the role of the Opnod1, Opnod2, and Optbk1 genes in antiviral and antibacterial immunity of spotted knifejaw (Oplegnathus punctatus). The expression patterns of these genes were analyzed using qRT-PCR in different tissues and at different time points. The open reading frame (ORF) of the Opnod1 gene was 2757 bp in length and encoded 918 amino acids, the ORF of the Opnod2 gene was 2970 bp in length and encoded 990 amino acids, while the Optbk1 gene was 2172 bp in length and encoded 723 amino acids. The Opnod1 and Opnod2 proteins contained three conserved domains (CARD, NOD, and LRR), and Optbk1 contained an STKc domain. The Opnod1, Opnod2, and Optbk1 genes were mainly expressed in immune-related tissues of spotted knifejaw, with the highest relative expression of the Opnod1 in the skin, the Opnod2 in the gill, and the Optbk1 in the liver. The expression of these genes changed significantly in the immune tissues following infection with SKIV-SD and Vibrio harveyi. In kidney cells, the Opnod1, Opnod2, and Optbk1 expression was up-regulated after stimulation by poly I:C and LPS in vitro. The results suggest that the NOD1/2-TBK1 signal pathway may play an important role in the resistance of the spotted knifejaw to virus and bacteria, providing valuable insights for disease-resistant breeding.

Giant grouper (Epinephelus lanceolatus) is an important economical aquaculture species that suffers from severe outbreaks of infectious diseases caused by infectious viruses, such as iridovirus and nervous necrosis virus (NNV). To investigate the regulatory mechanisms of the antiviral immune response, we analysed the transcriptome profiling of the spleen and kidney of the giant grouper after spotted knifejaw iridovirus (SKIV) infection. We obtained a total of 3359 and 1294 differentially expressed genes (DEGs) in the spleen and kidney, respectively, among which 418 DEGs were shared between spleen and kidney. A total of 933 gene ontology (GO) terms and 27 significantly enriched GO terms (corrected p‐value < 0.05) were obtained in the spleen based on DEGs, and the corresponding values in the kidney were 720 and 3 respectively. GO analysis indicated that ‘metabolism’, ‘transmembrane transport’ and ‘binding’ occupied the dominant functional categories involved in the SKIV infection response. KEGG analysis revealed that the enriched pathways associated with several innate immune pathways, such as the Toll‐like receptor, RIG‐I‐like receptor and NOD‐like receptor signalling pathways, as well as gene expression associated with these pathways, were significantly affected in response to the SKIV infection. In addition, changes in the profiles of immune‐related gene expression from two highlighted pathways, namely the Toll‐like receptor signalling pathway and RIG‐I‐like receptor signalling pathway, were validated by qRT‐PCR.

FIGLA (factor in the germ line alpha) is a basic helix‐loop‐helix transcription factor specific in female germ cells and is essential for folliculogenesis in vertebrates. In this study, we cloned and characterized a figla gene from the turbot, Scophthalmus maximus. The full‐length cDNA of the turbot figla consists of 1,163 nucleotides, with a 603 bp open reading frame (ORF) encoding a 201‐ amino ‐acid protein. Genomic structure analysis and multiple protein sequence alignments showed a conserved exon‐intron organization and high sequence similarity among some flatfish species, which is divergent from other teleost. Quantitative real‐time PCR analysis revealed that in 8, 14 and 24 months posthatching (mph) fish, figla was abundantly expressed in ovary, but significantly lower expressed in testis. In the 24 mph turbot, figla was predominantly expressed in ovary and slightly expressed in female liver and heart, but was hardly detectable in all tested tissues of males. In addition, in situ hybridization demonstrated that figla expressed in oocytes at early vitellogenesis phase of gonadal germ cell, but marginally present in testis. Taken together, these results indicated that the turbot figla is a female‐predominantly expressed gene that may function in the maintenance and development of the ovary.