Functional analysis of the ube3a response in Japanese flounder (Paralichthys olivaceus) to CSBV infection.

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb, traf3, and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida.

Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZEp, was characterized in E. piscicida. hutZEp is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZEp is probably not related to hemin utilization. To explore the biological role of HutZEp, a markerless hutZEp in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZEp did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZEp deletion, which indicated that HutZEp was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZEp significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZEp was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZEp is essential for E. piscicida biofilm formation and contributes to host infection.

Population response of intestinal microbiota to acute Vibrio alginolyticus infection in half-smooth tongue sole (Cynoglossus semilaevis).

Introduction: Vibriosis causes enormous economic losses of marine fish. The present study investigated the intestinal microbial response to acute infection of half-smooth tongue sole with different-dose Vibrio alginolyticus within 72 h by metagenomic sequencing. Methods: The inoculation amount of V. alginolyticus for the control, low-dose, moderate-dose, and high-dose groups were 0, 8.5 × 101, 8.5 × 104, and 8.5 × 107 cells/g respectively, the infected fish were farmed in an automatic seawater circulation system under a relatively stable temperature, dissolved oxygen and photoperiod, and 3 ~ 6 intestinal samples per group with high-quality DNA assay were used for metagenomics analysis. Results: The acute infections with V. alginolyticus at high, medium, and low doses caused the change of different-type leukocytes at 24 h, whereas the joint action of monocytes and neutrophils to cope with the pathogen infection only occurred in the high-dose group at 72 h. The metagenomic results suggest that a high-dose V. alginolyticus infection can significantly alter the intestinal microbiota, decrease the microbial α-diversity, and increase the bacteria from Vibrio and Shewanella, including various potential pathogens at 24 h. High-abundance species of potential pathogens such as V. harveyii, V. parahaemolyticus, V. cholerae, V. vulnificus, and V. scophthalmi exhibited significant positive correlations with V. alginolyticus. The function analysis revealed that the high-dose inflection group could increase the genes closely related to pathogen infection, involved in cell motility, cell wall/ membrane/envelope biogenesis, material transport and metabolism, and the pathways of quorum sensing, biofilm formation, flagellar assembly, bacterial chemotaxis, virulence factors and antibiotic resistances mainly from Vibrios within 72 h. Discussion: It indicates that the half-smooth tongue sole is highly likely to be a secondary infection with intestinal potential pathogens, especially species from Vibrio and that the disease could become even more complicated because of the accumulation and transfer of antibiotic-resistance genes in intestinal bacteria during the process of V. alginolyticus intensified infection.

The Isolation and Full-Length Transcriptome Sequencing of a Novel Nidovirus and Response of Its Infection in Japanese Flounder (Paralichthys olivaceus).

A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder (Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events (12,352) than the R group (11,452). The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.

Toxicity of nickel and cobalt in Japanese flounder.

Nickel and cobalt are essential elements that become toxic at high concentrations. Little is known about nickel and cobalt toxicity in aquatic animals. This study aimed to investigate acute and chronic toxicity of nickel and cobalt in Japanese flounder (Paralichthys olivaceous), with emphasis on oxidative stress reactions, histopathological changes, and differences in gene expression. The lethal concentration for 50% mortality (LC50) in 3 and 8 cm Japanese flounder exposed to nickel for 96 h was found to be 86.2 ± 0.018 and 151.3 ± 0.039 mg/L; for cobalt exposure, LC50 was 47.5 ± 0.015 and 180.4 ± 0.034 mg/L, respectively. Chronic nickel and cobalt exposure caused different degrees of oxidative enzyme activity changes in gill, liver, and muscle tissues. Erythrocyte deformations were detected after acute or chronic exposure to nickel and cobalt. the nickel and cobalt exposure also caused pathological changes such as spherical swelling over other gill patches, rod-like proliferations in the gill patch epithelial cell layer, and disorder in hepatocyte arrangement, cell swelling, and cytoplasm loosening. RNA-Seq indicated that there were 184 upregulated and 185 downregulated genes in the liver of Japanese flounder exposed to 15 mg/L nickel for 28 d. For cobalt, 920 upregulated and 457 downregulated genes were detected. Among these differentially expressed genes, 162 were shared by both nickel and cobalt exposure. In both nickel and cobalt, pathways including fatty acid elongation, steroid biosynthesis, unsaturated fatty acid biosynthesis, fatty acid metabolism, PPAR signaling, and ferroptosis were significantly enriched. Taken together, these results aided our understanding of the toxicity of nickel and cobalt in aquatic animals.