Toxic Effects of Koumine on the Early-Life Development Stage of Zebrafish.

Koumine is one of the most abundant alkaloids found in Gelsemium elegans, and it has a wide range of pharmacological effects including antitumor, anti-inflammatory, analgesic treatment effects, and antianxiety. However, its high toxicity and unclear mechanism of action have greatly limited the medicinal development and use of koumine. We investigated the toxic effects of koumine on the developmental toxicity and behavioral neurotoxicity of zebrafish embryos and larvae. Embryos at 6 h postfertilization (hpf) were exposed to 12.5, 25, 50, 75, and 100 mg/L of koumine until 120 hpf. Koumine affected the hatching and heartbeats of the embryos. The morphological analysis also revealed many abnormalities, such as shortened bodies, yolk sac edemas, tail malformations, and pericardial edemas. To identify the neurotoxicity of koumine, the behavior of the larvae was measured. Koumine at 50 and 100 mg/L affect the escape response. The embryos exhibited uncoordinated muscle contractions along the body axis in response to touch at 36 hpf. More importantly, we found that the neurotoxicity of koumine is mainly caused by influencing the ACh content and the activity of AChE without impairing motor neuron development. A comprehensive analysis shows that a high concentration of koumine has obvious toxic effects on zebrafish, and the safe concentration of koumine for zebrafish should be less than 25 mg/L. These results will be valuable for better understanding the toxicity of koumine and provide new insights into the application of koumine.

mitfa deficiency promotes immune vigor and potentiates antitumor effects in zebrafish.

The mitfa gene is a well-known transcription factor associated with microphthalmia and is essential for early melanophore development. However, little is known about how mitfa affects the immune system. Here, we generated a novel mitfa knock-out zebrafish line using the CRISPR/Cas9 system. The mitfa-/- zebrafish exhibited reduced melanin levels compared to the nacre mutant. We investigated the impact on the immune system after exposure to Edwardsiella tarda and bifenazate in zebrafish larvae, and observed that the macrophage numbers were reduced in both treated groups. Remarkably, the expression levels of immune-related genes exhibited significant increases after bacterial challenge or bifenazate exposure in the mitfa-/- zebrafish, except for tlr4 and rela. Furthermore, we conducted xenograft experiments using mouse B16 melanoma cells. Notably, the cancer cells didn't show a high cell migration ratio, implying that the immune system was highly activated after the loss of mifta. Taken together, our findings suggest that mitfa-/- zebrafish serve as a valuable model for investigating the relationship between the immune system and melanocytes, providing new insights into the role of mitfa in immune responses.

Single and Combined Effects of Microplastics and Cadmium on the Cadmium Accumulation and Biochemical and Immunity of Channa argus.

Microplastics can accumulate residual drugs and heavy metals in the environment and accumulate through the layers of the food chain, ultimately causing harm to human health. The pollution of microplastics in the freshwater environment is becoming more and more serious, which directly affects the safety of aquatic organisms. This experiment studied the effects of single and composite microplastics and Cd on the tissue damage, antioxidant, and immune response of juvenile Channa argus. Microplastics with different diameters of 80 nm, 0.5 µm (200 µg/L), and Cd (50 µg/L) were used for exposure, and four sampling points were set for 24 h, 48 h, 96 h, and clear 48 h. Under different treatments, a certain degree of gill tissue damage can be found in 96 h. Microplastics and Cd can cause oxidative stress and affect the antioxidant status, and the impact of 0.5-µm microplastics is stronger than that of 80-nm microplastics. There is an antagonistic effect between the two microplastics and Cd during compound exposure, but the activity of CAT shows a synergistic effect. Microplastics and Cd affect the expression of immune-related genes to varying degrees. When exposed together, the expression of HSP70 gene all showed mutual antagonism, while the expression of IL-1ß gene was different. The expression of the MT gene can infer the ability of microplastics to accumulate Cd, and microplastics with a small diameter of 80 nm have stronger enrichment capabilities.

Effects of mixed antimicrobial peptide on the growth performance, antioxidant and immune responses and disease resistance of Pengze crucian carp (Carassius auratus var. Pengze).

Antimicrobial peptides have broad-spectrum antibacterial properties and low drug resistance, and they demonstrate great potential as antibiotic substitutes. In this study, five dietary mixed antimicrobial peptide supplement groups were set and fed to Pengze crucian carp for 10 weeks. The 6 groups were G0 (control group) and 5 additional groups: G1 (100 mg/kg), G2 (200 mg/kg), G3 (400 mg/kg), G4 (800 mg/kg) and G5 (1600 mg/kg). The results showed that the final body weight (FBW), weight gain rate (WGR) and specific growth rate (SGR) of fish in G1 and G2 were higher than those of fish in the control group, and G1 was significantly higher than G0 (P < 0.05). In addition, the FBW, WGR, and SGR of the G3 group were significantly lower than those of the G0 group. The chymotrypsin, lipase and amylase activities of G1 and G2 were significantly upregulated compared with G0 and reached peak values in G1. The activity of T-AOC and SOD in the addition group was higher (except G2 and G4) than that in the control groups, and significantly increased in G3 compared to the control group. The activity of MDA in the addition group was lower than that in the control group (p > 0.05). The expression levels of TLR-4, MYD88 and TNF-α in the three organs of the addition group were higher than those in G0 and reached the peak value in G3 (p < 0.05). Furthermore, the expression levels of TLR-4, MYD88 and TNF-α in the three organs of G3 were significantly lower than those in G0 and lower than those in the other supplemented groups. The expression levels of IL-10 and IL-11 tended to be upregulated after A. hydrophila challenge, and G3 in different organs was significantly higher than that in other supplemented groups and G0. The results of this study show that an appropriate amount of mixed antimicrobial peptides can improve the growth performance and antioxidant and immune capabilities of Pengze crucian carp and can also play a positive role in the treatment of A. hydrophila infection.

Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish.

The molecular pathogenesis of glioblastoma indicates that RTK/Ras/PI3K, RB and TP53 pathways are critical for human gliomagenesis. Here, several transgenic zebrafish lines with single or multiple deletions of nf1, tp53 and rb1 in astrocytes, were established to genetically induce gliomagenesis in zebrafish. In the mutant with a single deletion, we found only the nf1 mutation low-efficiently induced tumour incidence, suggesting that the Nf1 pathway is critical for the initiation of gliomagenesis in zebrafish. Combination of mutations, nf1;tp53 and rb1;tp53 combined knockout fish, showed much higher tumour incidences, high-grade histology, increased invasiveness, and shortened survival time. Further bioinformatics analyses demonstrated the alterations in RTK/Ras/PI3K, cell cycle, and focal adhesion pathways, induced by abrogated nf1, tp53, or rb1, were probably the critical stepwise biological events for the initiation and development of gliomagenesis in zebrafish. Gene expression profiling and histological analyses showed the tumours derived from zebrafish have significant similarities to the subgroups of human gliomas. Furthermore, temozolomide treatment effectively suppressed gliomagenesis in these glioma zebrafish models, and the histological responses in temozolomide-treated zebrafish were similar to those observed in clinically treated glioma patients. Thus, our findings will offer a potential tool for genetically investigating gliomagenesis and screening potential targeted anti-tumour compounds for glioma treatment.

Benzo[a]pyrene induces microbiome dysbiosis and inflammation in the intestinal tracts of western mosquitofish (Gambusia affinis) and zebrafish (Danio rerio).

Benzo[a]pyrene (BaP) is one of the most well studied carcinogenic polycyclic aromatic hydrocarbons (PAHs) that has been associated with a wide range of toxic effects in aquatic organisms. In the present study, the mosquitofish and zebrafish were exposed to BaP (100 µg/L) for 15 days. We analyzed the intestinal microbial community of mosquitofish and zebrafish using 16S rRNA gene amplicon sequencing and also performed transcriptional profiling of the inflammation pathway related genes in the intestinal tissues. Our results showed that BaP exposure induced similar changes to the composition of microbial community in mosquitofish and zebrafish. At the phylum level, the abundance of Proteobacteria decreased while the abundance of Firmicutes increased following BaP exposure. At the genus level, a common pathogenic genus staphylococcus significantly increased in the BaP treatment groups, compared to the control (DMSO, ~0.001% v/v). In addition, it was observed that BaP significantly increased the mRNA level of il1ß in both mosquitofish and zebrafish. The transcript levels of il6, il8, il10 and ifnphi1 were significantly increased in zebrafish, however not in mosquitofish, following Bap exposure. Our findings suggest that BaP could induce microbiota dysbiosis and inflammation in the intestine of mosquitofish and zebrafish.

Expression of HSP70 family mRNAs in albino northern snakehead, Channa argus: Response to extreme temperature stress and bacterial infection.

The wild albino northern snakehead, Channa argus, has only been found in Jialing Rivers System of China so far. It is easy to be affected by the environmental factors such as temperature changes and bacterial infection, thus causing a huge economic loss. Therefore, this study cloned a 2,213 bp cDNA that encodes a protein of heat shock protein 70 (CaHSP70), which has an open reading frame (ORF) that encodes 639 amino acids and the corresponding polypeptides of 70.50 kDa. And the oretical isoelectric point (pI) of CaHSP70 is 5.79. Additionally, we also cloned a cDNA for heat shock cognate protein 70 (CaHSC70) with a total length of 2,300 bp. And the ORF of CaHSC70 encodes 648 amino acids and 71.18 kDa peptides. The pI of CaHSC70 is 5.22. Moreover, the cDNA length of stress-70 protein mitochondrial (CaHSPA9) is 2,944 bp with an ORF that encodes 679 amino acids, polypeptides of 73.74 kDa, and a pI of 6.68. The quantitative real-time PCR (qRT-PCR) analysis showed that the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 genes were tissue-specific in the control groups. After the heat shock at 37 °C, the mRNA expression levels of CaHSP70 were extremely significantly upregulated in the kidney, liver, spleen, and brain tissues, while fewer mRNA expression levels of CaHSC70 and CaHSPA9 showed a relatively induction in these tissues. In contrast, after the cold shock at 8.5 °C, fewer mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 showed the changes of expression in all the tissues, compared to heat shock. In addition, CaHSP70, CaHSC70, and CaHSPA9 mRNA expression levels showed an overall trend of first upregulating and then falling after Edwardsiella tarda (strain No. DL1,476) challenge. In conclusion, this study demonstrated that temperature had a great effect on the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9, and the mRNA expression levels of these three genes were also sensitive to pathogen infection, especially CaHSP70 in the albino C. argus.

Effects of dietary Gelsemium elegans alkaloids on intestinal morphology, antioxidant status, immune responses and microbiota of Megalobrama amblycephala.

Numerous plant extracts used as feed additives in aquaculture have been shown to stimulate appetite, promote growth and enhance immunostimulatory and disease resistance in cultured fish. However, there are few studies on the famous Chinese herbal medicine Gelsemium elegans, which attracts our attention. In this study, we used the Megalobrama amblycephala to investigate the effects of G. elegans alkaloids on fish intestinal health after diet supplementation with 0, 5, 10, 20 and 40 mg/kg G. elegans alkaloids for 12 weeks. We found that dietary G. elegans alkaloids at 40 mg/kg improved intestinal morphology by increasing villus length, muscle thickness and villus number in the foregut and midgut and muscle thickness in the hindgut (P < 0.05). These alkaloids also significantly improved intestinal antioxidant capabilities by increasing superoxide dismutase, catalase, total antioxidant capacity and malondialdehyde levels and up-regulated intestinal Cu/Zn-SOD and Mn-SOD (P < 0.05) at 20 and 40 mg/kg. Dietary G. elegans alkaloids improved intestinal immunity via up-regulating the pro-inflammatory cytokines IL-1ß, IL-8, TNF-α and IFN-α and down-regulating expression of the anti-inflammatory cytokines IL-10 and TGF-ß (P < 0.05) at 20 and 40 mg/kg. The expression of Toll-like receptors TRL1, 3, 4 and 7 were also up-regulated in intestine of M. amblycephala (P < 0.05). In intestinal microbiota, the abundance of Proteobacteria was increased while the Firmicutes abundance was decreased at phylum level after feeding the alkaloids (P < 0.05). The alkaloids also increased the abundance of the probiotic Rhodobacter and decreased the abundance of the pathogenic Staphylococcus at genus level (P < 0.05). In conclusion, dietary G. elegans alkaloid supplementation promoted intestine health by improving intestine morphology, immunity, antioxidant abilities and intestinal microbiota in M. amblycephala.

Effects of fasting and re-feeding on mstn and mstnb genes expressions in Cranoglanis bouderius.

The myostatin (mstn) and myostatinb (mstnb) gene of Cranoglanis bouderius were cloned and sequenced and their expressions under nutritional restriction were characterized. The full cDNA sequences of mstn and mstnb were 1878 bp and 1928 bp, containing an open reading frame of 1170 bp and 1119 bp, which encoded 390 and 373 amino acids, respectively. The deduced mstn and mstnb sequence structures were similar to other members of TGF-ß superfamily, including the TGF beta pro-peptide, TGF beta domain, proteolytic processing site and nine conserved cysteines in the C-terminal. In addition, four mstn gene duplications were found in Cranoglanis bouderius. Sequence alignment and phylogenetic tree analyses indicated that the mstn gene and mstnb gene had a close relationship with Siluriformes fish, and the mstn and mstnb genes were roughly classified into two groups. RT-PCR analysis revealed that the mstn and mstnb were expressed in a variety of tissues in Cranoglanis bouderius although the mstn was highly expressed in skeletal muscle and the mstnb was mainly expressed in brain. We speculate that the mstn gene but not mstnb is likely to play a key role in managing muscle growth. A fasting-re-feeding experiment was used to evaluate the effects of starvation on mstn and mstnb expressions in juvenile Cranoglanis bouderius for 5 weeks. The result showed that the mstn and mstnb transcript levels varied among tissues. The mRNA expression levels of mstn in muscle, brain and liver gradually decreased during starvation and returned to the normal level after re-feeding. The mstnb mRNA levels in muscle, brain, liver, spleen, intestine and kidney increased during an early fast time but ultimately decreased with prolonged fasting time. The mstnb transcript levels in muscle, brain and liver increased significantly after re-feeding. In summary, the results supported that the mstn and mstnb may not be limited to control of muscle growth in fish but could also be involved in other biological functions.

HSP60 expression profile under different extreme temperature stress in albino northern snakehead, Channa argus.

The great albino northern snakehead, Channa argus, is one of the most important economical fish in China. In the present study, cDNA encoding heat shock protein 60 (HSP60) was cloned and characterized. The cDNA was 2462 bp, containing an open reading frame (ORF) encoding a 575-amino-acids polypeptide of 61.10 kDa (theoretical isoelectric point [pI]: 5.66). BLAST analysis showed that AcaHSP60 was highly similar with other HSP60s, and three conserved amino acid blocks and characteristic motifs or domains defined as HSP60 protein family signatures. Genomic DNA analysis showed that AcaHSP60 had ten exons in the coding region (from 94 to 336 bp). Changes in AcaHSP60 gene expression profiles in albino C. argus experimentally exposed to different temperature stress (8.5, 26, and 37 °C) was investigated. Quantitative real-time PCR and western blot analysis revealed that tissue-specific AcaHSP60 expressions were in the spleen, muscle, liver, kidney, heart and brain. Expression was highly significantly stimulated after heat shock (37 °C), but showed no significant differences after cold treatment (8.5 °C) except in the brain. In summary, these results showed that AcaHSP60 was significantly tissue specific and indicate that AcaHSP60 expression might be sensitive to thermal resistance in albino C. argus.

Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant (POP), an unintentional byproduct of various industrial processes, and a human carcinogen. The expression of the cytochrome P450 1A (cyp1a) gene is upregulated in the presence of TCDD through activating the aryl hydrocarbon receptor pathway in a dose-dependent manner. Several essential response elements, including the 8 potential xenobiotic response elements in the cyp1a promoter region, have been identified to be the main functional parts for the response to TCDD. Thus, we aimed to develop a convenient and sensitive biomonitoring tool to examine the level of POPs in the environment and evaluate its potential human health risks by TCDD. Here, we established a transgenic zebrafish model with a red fluorescent reporter gene ( mCherry) using the truncated cyp1a promoter. Under exposure to TCDD, the expression pattern of mCherry in the reporter zebrafish mirrored that of endogenous cyp1a mRNA, and the primary target tissues for TCDD were the brain vessels, liver, gut, cloaca, and skin. Our results indicated that exposure of the embryos to TCDD at concentrations as low as 0.005 nM for 48 h, which did not elicit morphologic abnormalities in the embryos, markedly increased mCherry expression. In addition, the reporter embryos responded to other POPs, and primary liver cell culture of zebrafish revealed that Cyp1a protein was mainly expressed in the cytoplasm of liver cells. Furthermore, our transgenic fish embryos demonstrated that TCDD exposure can regulate the expression levels of several tumor-related factors, including epidermal growth factor, TNF-α, C-myc, proliferating cell nuclear antigen, TGF-ß, serine/threonine kinase (Akt), and phosphorylated Akt, suggesting that our transgenic fish can be used as a sensitive model to evaluate the carcinogenicity induced by TCDD exposure.-Luo, J.-J., Su, D.-S., Xie, S.-L., Liu, Y., Liu, P., Yang, X.-J., Pei D.-S. Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

Generation and application of a novel transgenic zebrafish line Tg(cyp1a:mCherry) as an in vivo assay to sensitively monitor PAHs and TCDD in the environment.

The polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) are classified as human carcinogens, and can also cause serious health problems. To develop a convenient bio-monitoring tool for the detection of PAHs and TCDD in the environment, we generated a transgenic zebrafish line Tg(cyp1a:mCherry) with cyp1a promoter driving mCherry expression. Here, Tg(cyp1a:mCherry) embryos were treated with different concentrations of TCDD and five US EPA priority PAHs congeners. The results showed that the expressions of mCherry and endogenous cyp1a were consistent with the PAHs exposure concentrations and were largely induced by TCDD and ≥4-ring PAHs. Moreover, the sensitivity of Tg(cyp1a:mCherry) embryos was also evaluated through monitoring of the PAHs contamination in the water and soil samples. The elevated red fluorescent signals and cyp1a expression levels were observed in Tg(cyp1a:mCherry) zebrafish after exposure to water samples and soil organic extracts with higher concentrations of ≥4-ring PAHs. These results further strengthen our findings of concentration- and congener-dependent response of the newly established zebrafish. Taken together, the newly established zebrafish line will prove as a sensitive, efficient and convenient tool for monitoring PAHs and TCDD contamination in the environment.

Molecular cloning, characterization and expression analysis of heat shock protein 90 in albino northern snakehead Channa argus.

The great albino northern snakehead Channa argus is habitual to only the Sichuan Jialing Rivers System in China, making its introduction difficult to other riverine systems. Here, we characterized heat shock protein 90 (AcaHSP90) and probed its molecular responses toward the environmental stressors that C. argus can face during its introduction and breeding in the other southern latitudes of China. To serve the purpose, cDNA encoding of AcaHSP90 were cloned and characterized in albino C. argus. The cDNA was 2752bps that contained an open reading frame (ORF), encoding a 726-amino-acid polypeptide of 83.35kDa (theoretical isoelectric point [pI]: 4.89). Genomic DNA analysis showed that the AcaHSP90 gene consisted of 7 introns, five conserved amino acid blocks and other motifs or domains. The AcaHSP90 structure was highly similar with the other known HSP90s except those identified in the bacteria. The expression profiles of AcaHSP90 gene in albino C. argus were also investigated after experimentally exposed to different temperature stresses (8.5, 26 and 37°C) and infected with Edwardsiella tarda (strain NO. DL1476) at different time intervals (0, 6, 12, 24, 36, 48, 72h). In addition, the AcaHSP90 expression in different tissues of albino C. argus were also analyzed. The quantitative real-time PCR and western blot analysis revealed tissue-specific AcaHSP90 expressions in control group, and expressions were significantly stimulated in the brain, heart, kidney, liver, muscle and spleen after the heat shock (37°C), while showed no significant difference after the cold treatment (8.5°C). The mRNA levels of AcaHSP90 were also significantly upregulated in the spleen and muscle at 12h and in the kidney at 12 and 48h post pathogen injections. In a nut shell, these novel results showed tissue-specific responses of AcaHSP90 and indicated that this heat shock protein might also be sensitive to pathogen infection, but closely related to the thermal resistance in albino C. argus.