Analysis of the immune function of Caspase-3 in Cristaria plicata.

Caspase-3 is generally considered to be the most important terminal shear enzyme in the process of apoptosis, as well as an important part of cytotoxic T lymphocytes (CTL) killing mechanism, which is confirmed to play an important role in vertebrate cell apoptosis and immune system, and is poorly reported in invertebrates. In this paper, we used bioinformatics to perform amino acid multiple sequence alignment and protein structural domain analysis, and constructed a phylogenetic tree to identify the full-length cDNA of the cloned caspase-3 of Cristaria plicata (Named CpCaspase-3). The expression of caspase-1, caspase-7, caspase-8, and caspase-9 was found to be down-regulated by double-stranded RNA interference of CpCaspase-3 in C. plicata. Some degree of disruption of the caspase signaling pathway occurs. The expression of CpCaspase-3 was affected after injection of Lipopolysaccharide (LPS), Peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly(I:C)), and Aeromonas hydrophila. These results were suggested that CpCaspase-3 was involved in the immune response of C. plicata. The wound recovery process of C. plicata was simulated and CpCaspase-3 was found to promote wound recovery. An autophagy inhibition and autophagy activation model of mussels was constructed, where apoptosis and autophagy undergo crosstalk, and inhibition of autophagy induces the onset of apoptosis, and similarly autophagy activation inhibits the process of apoptosis instead. In addition, a recombinant CpCaspase-3-pEGFP-C1 plasmid was constructed for subcellular localization experiments and found that CpCaspase-3 was distributed in both the nucleus and the cytoplasm. This paper aims to unveil the immune mechanism of C. plicata and provide a theoretical basis for the healthy culture of shellfish.

Immunotoxicity and transcriptome analysis of zebrafish embryos exposure to Nitazoxanide.

Nitazoxanide (NTZ) is a broad-spectrum immunomodulatory drug, and little information is about the immunotoxicity of aquatic organisms induced by NTZ. In the present study, reduced body length and decreased yolk sac absorption in the NTZ-treated group were observed. Meanwhile, the number of innate immune cells and adaptive immune cells was substantially reduced upon NTZ exposure, and the migration and retention of macrophages and neutrophils in the injured area were inhibited. Following NTZ stimulation, oxidative stress levels in the zebrafish increased obviously. Mechanistically, RNA-seq, a high-throughput method, was performed to analyze the global expression of differentially expressed genes (DEGs) in zebrafish embryos treated with NTZ. 531 DEGs were identified by comparative transcriptome analysis, including 121 up-regulated and 420 down-regulated genes in zebrafish embryos after NTZ exposure. The transcriptome sequences were further subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) and analysis, showing phototransduction and metabolic pathway, respectively, and were most enriched. In addition, some immune-related genes were inhibited after NTZ exposure. RNA-seq results confirmed by qRT-PCR were used to verify the expression of the 6 selected genes. The other immune-related genes such as two pro-inflammatory cytokines (IL-1ß, tnfα) and two chemokines (CXCL8b.3, CXCL-c1c) were further confirmed and were differentially regulated after NTZ exposure. In summary, NTZ exposure could lead to immunotoxicity and increased ROS in zebrafish embryos, this study provides valuable information for future elucidating the molecular mechanism of exogenous stimuli-induced immunotoxicity in aquatic ecosystems.

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress.

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at -440 bp to +54 bp, and that of PcGSTD2 promoter was between -1609∼-1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

The thioredoxin expression of Cristaria plicata is regulated by Nrf2/ARE pathway under microcystin stimulation.

Thioredoxin plays an important role in inhibiting apoptosis and protecting cells from oxidative stress. This study was aimed to clarify how the expression of Trx from Cristaria plicata is regulated by Nrf2/ARE pathway. The expression of CpTrx mRNA was significantly up-regulated in gill and kidney tissues under microcystin stress. The Nrf2 gene of Cristaria plicata was identified to possess an auto active domain bit. While CpNrf2 was knocked down by specific small RNA, CpTrx mRNA expression was significantly down-regulated. The promoter of CpTrx gene had high transcriptional activity, and this basic transcriptional activity persisted after ARE element mutation. The region of promoter -206 to +217 bp was a core promoter region and had forward regulatory elements. Gel shift Assay exhibited that the CpTrx promoter could bind to the purified proteins CpNrf2 and CpMafK in vitro. The binding phenomenon disappeared after the ARE element mutation in promoter region. Subcellular localization experiments displayed that fluorescence overlap between CpNrf2 and Trx promoter increased under microcystin toxin stress. These results suggested that Trx expression was regulated by Nrf2/ARE pathway under oxidative stress.

Insights into the circadian rhythm alterations of the novel PFOS substitutes F-53B and OBS on adult zebrafish.

As alternatives to perfluorooctane sulfonate (PFOS), 6:2 Cl-PFESA (F-53B) and sodium p-perfluorous nonenoxybenzene sulfonate (OBS) are frequently detected in aquatic environments, but little is known about their neurotoxicity, especially in terms of circadian rhythms. In this study, adult zebrafish were chronically exposed to 1 µM PFOS, F-53B and OBS for 21 days taking circadian rhythm-dopamine (DA) regulatory network as an entry point to comparatively investigate their neurotoxicity and underlying mechanisms. The results showed that PFOS may affect the response to heat rather than circadian rhythms by reducing DA secretion due to disruption of calcium signaling pathway transduction caused by midbrain swelling. In contrast, F-53B and OBS altered the circadian rhythms of adult zebrafish, but their mechanisms of action were different. Specifically, F-53B might alter circadian rhythms by interfering with amino acid neurotransmitter metabolism and disrupting blood-brain barrier (BBB) formation, whereas OBS mainly inhibited canonical Wnt signaling transduction by reducing cilia formation in ependymal cells and induced midbrain ventriculomegaly, finally triggering imbalance in DA secretion and circadian rhythm changes. Our study highlights the need to focus on the environmental exposure risks of PFOS alternatives and the sequential and interactive mechanisms of their multiple toxicities.

Prx5 of Cristaria plicata has antioxidant function and is regulated by Nrf2/ARE signaling pathway.

Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.

Effects of sulfometuron-methyl on zebrafish at early developmental stages.

Sulfometuron methyl (SM) is a widely used herbicide and thus leading to accumulation in the environment. The toxicity assessments of SM in model organisms are currently rare. In the present study, zebrafish were utilized for evaluating the detrimental effects of SM in aquatic vertebrates. Zebrafish embryos were exposed to 0, 10, 20, and 40 mg/L SM from 5.5 to 72 h post-fertilization (hpf), respectively. Consequently, SM exposure resulted in increasing the mortality rate and reducing hatching rate in larval zebrafish at 10, 20, and 40 mg/L SM-treated groups. The reduced numbers of immune cells (neutrophils and macrophages) were observed after SM exposure by a dose-dependent manner. The inflammatory responses (TLR4, MYD88, IL-1ß, IL-6, IL-8, IFN-γ, IL-10, and TGF-ß) were measured to estimate immune responses. Anti-inflammatory factors (IL-10 and TGF-ß) were down-regulated in all the treated groups and significantly altered at 40 mg/L exposure group. Additionally, behavioral tests suggested that SM treatment significantly increased the total distance, average speed, and maximum acceleration of larval zebrafish during light-dark transition and subsequently enzymology test displayed the same trend to locomotor behaviors. The content significantly increased in oxidative stress, as reflected in ROS level in all the treated groups. The numbers of cell apoptosis were significantly increased at 20, and 40 mg/L and the highest concentration group induced the substantial increment (P < 0.001) of apoptosis-related genes including p53, Bax/Bcl-2, caspase-9, and caspase-3. In summary, our results demonstrated that exposure to SM caused toxicity of development, immune system, locomotor behavior, oxidative stress, and cell apoptosis at the early developmental stages of zebrafish.