Jichang Jian

Trachinotus ovatus (golden pompano) represents one of the dominant high-value mariculture fish species farmed across coastal regions of China. In intensive farming modes, intestinal damage and immune suppression are frequent issues in golden pompano; the widespread application of antibiotics in disease control has also triggered increasing worries about ecological risks and aquatic product safety. Host-origin probiotics have been widely recognized as a green and safe strategy to mitigate health risks and promote growth in farmed fish. Nevertheless, the regulatory roles and mechanisms of autochthonous E. faecalis strains on the intestinal health of golden pompano are still poorly understood. In this study, a 42-day feeding trial was conducted using golden pompano reared in a large-capacity floating net-cage system. The experimental fish were fed diets supplemented with different concentrations of E. faecalis SZ07 (1 × 10⁵ to 1 × 10⁹ CFU/g feed). Dietary supplementation with SZ07 at suitable doses (1 × 10⁷ to 1 × 10⁸ CFU/g feed) notably improved intestinal tissue structure, elevated the activities of digestive, antioxidant, and immune enzymes, and modulated the mRNA levels of key immune genes in the spleen. Analysis of the gut microbiota revealed that dietary E. faecalis SZ07 reshaped the intestinal microbial community, with an increased abundance of beneficial taxa and a decreased proportion of opportunistic pathogens. Taken together, our results indicate that autochthonous E. faecalis SZ07 acts as an effective probiotic to enhance intestinal health and immune function of golden pompano and offers technical support for the healthy development of its aquaculture industry.

Non-specific cytotoxic cells (NCCs) are critical first-line defenses against bacterial pathogens in teleosts, yet the molecular mechanisms governing their activity remain poorly understood. This study characterized LOC109197777, a novel long non-coding RNA (lncRNA) from Nile tilapia (Oreochromis niloticus), as a positive regulator of NCC-mediated immunity against Streptococcus agalactiae. Bioinformatic and experimental analyses confirmed its non-coding potential and predominant cytoplasmic localization. In vitro, overexpression of LOC109197777 in tilapia head kidney cells upregulated NCC activation receptors while downregulating effector molecules and pro-inflammatory cytokines, indicating a "primed but restrained" activation state; conversely, siRNA-mediated knockdown reduced NCC cytotoxicity. In vivo, intraperitoneal delivery of LOC109197777 significantly enhanced survival following S. agalactiae challenge and reduced bacterial burdens in the head kidney and spleen. Furthermore, tissue-specific expression profiling revealed that LOC109197777 orchestrates spatially distinct regulatory programs: rapid activation in the head kidney, sustained responses in the spleen, early responses in the liver, and prolonged mucosal immunity in the intestine. Notably, the lncRNA differentially regulated perforin-1 and granzyme B in a tissue and time-dependent manner, balancing effective pathogen clearance with immune homeostasis. These findings establish LOC109197777 as the first characterized lncRNA regulator of NCC activity in teleosts, operating through a tissue-specific, temporally coordinated mechanism. This study provides a novel molecular target for genetic enhancement of disease resistance in tilapia aquaculture and offers a theoretical foundation for developing lncRNA-based immunomodulatory strategies.

Ammonia exposure can induce oxidative stress in aquatic animals. The p62 protein is a selective autophagy receptor that participates in protein degradation and oxidative stress regulation. In this study, the role of Lv-p62 in the response of Litopenaeus vannamei to ammonia exposure was investigated using RNA interference. The results showed that Lv-p62 expression was significantly induced in the hepatopancreas, gills, and intestine of L. vannamei after ammonia exposure (p < 0.05). Lv-p62 expression peaked at 6 h in the gills and 24 h in the intestine, whereas a biphasic response was observed in the hepatopancreas, with an initial peak at 12 h and a higher second peak at 48 h. In the RNAi experiment, Lv-p62 knockdown altered the expression of antioxidant-related genes (Trx, Gst, and Gpx) in a tissue-specific manner, with Gpx expression being prominently increased in the gills and intestine but not in the hepatopancreas under ammonia exposure. Autophagy-related genes (ATG4 and ATG10) also showed time-dependent and tissue-specific expression changes after Lv-p62 knockdown. The expression of apoptosis-related genes, including caspase 3 and p53, was tissue-specific and was generally lower in the dsRNA-Lv-p62+NH₃ group than in the dsRNA-EGFP+NH₃ group at most time points. Histopathological observations showed that hepatopancreatic acinar vacuolation and structural damage were alleviated, and the hepatopancreatic apoptosis rate was reduced in L. vannamei in the dsRNA-Lv-p62+NH₃ group. These findings suggest that Lv-p62 participates in the response of L. vannamei to ammonia exposure, possibly by regulating antioxidant defense, autophagy-related processes, and apoptosis, thereby affecting hepatopancreatic oxidative damage and tissue injury.

Thioredoxin (TRX) is an important redox-related protein, which plays a key role in maintaining redox balance in cells. However, the role of TRX in ammonia exposure of Oreochromis niloticus is still unknown. In this study, we successfully cloned the TRX gene (OnTRX) of O. niloticus and performed systematic bioinformatics analysis. The results of multi-sequence alignment and phylogenetic tree analysis showed that OnTRX is highly conserved in vertebrates, but exhibits low sequence similarity with TRX homologs from arthropods (crustaceans). The tissue distribution of OnTRX and its transcriptional response to ammonia challenge were determined by qRT-PCR, and we further investigated the relationship between OnTRX and the response of the Nile tilapia epithelial cell line (TSE-04) to NH₄Cl treatment. The results showed that OnTRX exhibited tissue-specific expression, with a relatively high expression level in gill tissue. Ammonia exposure could significantly induce the expression of OnTRX in the gill, head kidney, intestine, skin, liver, and spleen of tilapia. In TSE-04 cells, OnTRX overexpression was associated with reduced NH₄Cl-induced morphological damage, a lower proportion of apoptotic cells, and altered transcript levels of several stress-related genes. Collectively, these findings indicate that OnTRX is likely involved in the regulatory response of Nile tilapia to ammonia-induced stress, while the underlying molecular mechanisms thereof remain to be further elucidated.

Although numerous studies have focused on the potential application of heterotrophic nitrification–aerobic denitrification (HNAD) bacteria in wastewater treatment, research exploring their potential in aquaculture biofloc systems remains limited. In this study, a promising HNAD strain, identified as Stutzerimonas stutzeri MJ20, was isolated from mature biofloc. This strain efficiently utilized low-cost carbon sources (e.g., glucose) and small-molecule carbon sources (e.g., sodium acetate and sodium succinate). Under conditions with glucose as the carbon source, a carbon-to-nitrogen (C/N) ratio of 15, pH 6–9, temperature 25–35 °C, salinity 0–35‰, and shaker speed of 0–150 rpm, it achieved removal rates of 95–100% for NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N at initial concentrations of 100 mg/L each. Even at higher concentrations (up to 200 mg/L NH₄⁺-N and 500 mg/L for both NO₂⁻-N and NO₃⁻-N), removal rates exceeded 99%. Under mixed nitrogen sources, strain MJ20 demonstrated efficient nitrogen removal, preferentially utilizing NH₄⁺-N, with only minimal and transient accumulation of nitrite and nitrate. Genomic analysis revealed that MJ20 carries key denitrification genes, including napA, nirS, norB and nosZ, and possesses complete pathways for nitrate reduction to nitrogen gas and ammonia assimilation, although typical autotrophic nitrification genes were not detected. Combined genomic data and autotrophic culture experiments indicated that, in addition to utilizing various organic carbon sources, the strain also exhibited certain autotrophic growth capabilities. Furthermore, MJ20 showed strong flocculation ability (flocculation rate > 96% within 16 h), sensitivity to multiple common antibiotics, and no toxicity to zebrafish, demonstrating favorable biosafety. In simulated seawater aquaculture wastewater with a C/N ratio of 5, it achieved a total nitrogen removal rate exceeding 94% within 72 h. These results indicate that strain MJ20 possesses comprehensive advantages, including efficient nitrogen removal, broad carbon source adaptability, strong environmental resilience, minimal accumulation of intermediate nitrogen products, excellent flocculation ability, and high biosafety. These traits highlight its potential for application in biofloc systems and in treating aquaculture tail water with a low C/N ratio. This study provides theoretical insights and practical guidance for screening HNAD bacteria suitable for biofloc systems.

This study aimed to elucidate the key characteristics of the humoral immune response and the tissue distribution of specific antibody-secreting cells (ASCs) in Nile tilapia (Oreochromis niloticus). A specific immune model was established by immunizing fish with human IgG. Lymphocytes were isolated from the head kidney, spleen, and peripheral blood and subjected to antigen stimulation in vitro. The MTT assay, reflecting cell metabolic activity and viability, identified the optimal culture conditions as a cell concentration of 2.5 × 10⁶ cells/mL, an antigen concentration of 2 μg/mL, and a culture duration of 72 h. Under these conditions, peripheral blood lymphocytes exhibited the most increase in metabolic activity, followed by head kidney lymphocytes, while splenic lymphocytes showed no significant response. Subsequent dynamic monitoring of antibody-secreting cells (ASCs) using ELISPOT revealed that, in the absence of antigen stimulation, ASC numbers from all three tissues declined over time. Notably, head kidney ASCs retained approximately 50% of their initial number by day 5, whereas ASCs in peripheral blood and spleen decayed to barely detectable or completely undetectable levels, respectively. These findings suggest that the head kidney may serve as a primary site for ASC persistence during the effector phase, potentially contributing to sustained humoral immunity. Although antigen stimulation did not induce significant ASC expansion, it significantly slowed their decay rate (p < 0.05), indicating an antigen-dependent maintenance role. ELISA detection of antibody levels in the culture supernatants showed a consistent trend with the ELISPOT results, further confirming the sustained functional support of antigen for ASCs. Additionally, LPS stimulation experiments demonstrated that all three tissues contained plasmablasts activatable by non-specific stimuli, with peripheral blood showing the highest proliferation fold (4–6 times). In conclusion, this study provides insights into the tissue-specific distribution, in vitro persistence, and antigen-dependent maintenance of ASCs in Nile tilapia, providing insights into the cellular basis that may contribute to humoral immune memory and laying a theoretical foundation for the rational design and application of tilapia vaccines.

Research on heterotrophic bioflocs is extensive, whereas investigations into autotrophic bioflocs remain limited. This study established an in situ autotrophic biofloc (ABF) system for intensive Pacific white shrimp (Penaeus vannamei) farming, aiming for zero water exchange and optimized water quality. A 120-day indoor experiment tested three stocking densities (300 (T1), 250 (T2), and 200 shrimp per m³ (T3)) with no water exchange. Water quality was monitored every two days, and bacterial communities were analyzed on days 10 and 70. The results indicated that ABF maturation was achieved by day 70 across all treatments, marked by three key indicators: (1) synchronous declines in nitrite and nitrate concentrations; (2) concurrent decreases in pH and total alkalinity approaching maturation; and (3) sustained high nitrogen removal efficiency (nitrite < 0.7 mg/L, ammonia < 0.6 mg/L). All density groups displayed similar patterns in both water quality dynamics and microbial community evolution. Bacterial analysis revealed that dominant genera such as Ruegeria, Bacillus, Muricauda, SM1A02, and Nitrospira played critical roles in toxic nitrogen removal, while pathogenic Klebsiella and Vibrio significantly decreased post-maturation. Heterotrophic nitrification and aerobic denitrification microorganisms (HNADMs) were identified as potentially responsible for nitrite accumulation. Nitrite accumulation was found in all groups. T2 and T3 achieved satisfactory breeding performance despite pre-maturation nitrate peaks exceeding 40 mg/L, whereas T1 suffered a low survival rate (27.47%) due to severe nitrite accumulation (>50 mg/L). A biofloc volume (BFV) of 4–8 mL/L effectively managed daily feed inputs of 75–110 g/m³. These findings lay a theoretical and technical foundation for the application of in situ ABF cultivation in intensive farming and enhance the sustainability of aquaculture.

In mammals, CD22 is a member of the Siglec family and plays essential roles in B-cell activation, signal transduction, and immune regulation. However, its functions in teleost fish remain largely unclear. In this study, a CD22 homolog designated On-CD22 was identified and cloned from Nile tilapia (Oreochromis niloticus). On-CD22 transcripts were highly expressed in the head kidney and peripheral blood of healthy fish and showed significant expression changes following infection with Streptococcus agalactiae, Aeromonas hydrophila, or stimulation with poly(I:C). Subcellular localization analysis indicated that On-CD22 is predominantly localized to the plasma membrane. Luciferase reporter assays performed in heterologous cell systems showed that overexpression of On-CD22 was associated with changes in the basal transcriptional activities of NF-κB, IFN1, IFN3, and STAT1 responsive promoters under unstimulated conditions. Furthermore, single-cell transcriptomic analysis revealed that On-CD22 expression was mainly confined to the B-cell population within head-kidney leukocytes. Collectively, these findings suggest that On-CD22 may be involved in immune regulatory processes in Nile tilapia.

Hemolysin co-regulated protein 2 (Hcp2) is a core component of the type VI secretion system 2 in Vibrio alginolyticus, a widespread marine pathogen that infects humans and aquaculture species. Deletion of hcp2 (Δhcp2) significantly attenuated virulence in zebrafish larvae, showing reduced abdominal edema and impaired toll-like receptor (tlr)2 and tlr4-mediated innate immune activation compared with the wildtype strain. Enzyme-linked immunosorbent assay and cell lysis assays revealed that the Δhcp2 strain exhibited decreased levels of lipopolysaccharide (LPS) and peptidoglycan (PGN), along with increased cell permeability and abnormal cell wall structures observed by transmission electron microscopy. Proteomic and transcriptional analyses further demonstrated that expression of the Sec system components SecB, SecD, and SecF was markedly reduced in the Δhcp2 strain and positively regulated by hcp2. Bioinformatic prediction combined with protein–DNA docking analysis suggested that the transcription of secB, secD, and secF was co-regulated by the cAMP and cAMP Receptor Protein (cAMP–CRP) complex and RNA polymerase sigma D factor (RpoD), although rpoD expression itself remained unaffected. Together with previous evidence that Hcp2 positively regulates cAMP–CRP, these findings suggested that Hcp2 modulated LPS and PGN translocation, probably through the cAMP–CRP pathway, thereby maintaining cell wall integrity and virulence in V.alginolyticus.