Effect of aqueous extract of Millettia speciosa Champ on intestinal health maintenance and immune enhancement of Cyprinus carpio.

Millettia speciosa Champ (MSP) is a natural Chinese herb that improves gastrointestinal health and enhances animal immunity. An 8-week feeding trial with different MSP levels (0, 150, 300, and 600 mg/kg) was conducted to evaluate the promotive effects of MSP in Cyprinus carpio. Results indicate that MSP improved intestinal immunity to some extent evidenced by the immuno-antioxidant parameters and the 16S rRNA in the Illumina MiSeq platform. With the analysis of transcriptome sequencing, 4685 differentially expressed genes (DEGs) were identified, including 2149 up-regulated and 2536 down-regulated. According to the GO and KEGG enrichments, DEGs were mainly involved in the immune system. Transcriptional expression of the NOD-like signaling pathway and key genes retrieved from the transcriptome database confirmed that innate immunity was improved in response to dietary MSP administration. Therefore, MSP could be used as a feed supplement that enhances immunity. This may provide insight into Chinese herb additive application in aquaculture production.

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.

Generation and application of a novel transgenic zebrafish line Tg(GAcyp1a:eGFP/Luc) as an in vivo assay to sensitive and specific monitoring of DLCs in the environment.

CYP1A is the most commonly used biomarker and transgenic fish which carrying a cyp1a promoter to drive a reporter gene can be used as reliable way to monitor dioxin/dioxin-like compounds (DLCs) in the environment. Here, we cloned the cyp1a promoter of Gambusia affinis and this promoter showed stronger transcriptional activity than that of zebrafish. Then, a Tg(GAcyp1a:eGFP/Luc) transgenic zebrafish line was first constructed with the G. affinis cyp1a promoter driving eGFP expression using meganuclease I-SceI mediated transgenesis technology. The Tg(GAcyp1a:eGFP/Luc) larvae at 72 h post-fertilization (hpf) were tested by exposing to TCDD for 72 h, and induced GFP was mainly expressed in the liver with low background. The Tg(GAcyp1a:eGFP/Luc) zebrafish showed high sensitivity (limit of detection of 0.322 ng/L TCDD and 0.7 TEQ-ng/L PCDD/Fs) and specificity (insensitive to responses to PAHs and PCBs). In addition, the transgenic line showed a low detection concentration of the DLCs contaminated environmental samples (as low as 1.8 TEQ-ng/L), and the eGFP fluorescence intensity and the chemical-TEQ values were closely correlated. In conclusion, a sensitively and specifically transgenic zebrafish line was established to convenient and effective to detect DLCs in the environment.

Interactions effects of nano-microplastics and heavy metals in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

The interaction between microplastics and contaminants has potentially generated new undefined risks on animals and ecosystems, and nano-microplastics are considered to be more harmful than microplastics. This experiment investigated the interactions and effects of nano-microplastics with heavy metals cadmium in hybrid snakehead. Different concentrations of nano-microplastics 80 nm (50 µg/L and 500 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. Results indicated that the morphology of gill was altered under the influence of nano-microplastics and cadmium, and the damage was aggravated with time. Nano-microplastics and Cd can cause oxidative damage to fish liver partly by effect the activities of antioxidant enzyme, and significantly suppressed the expressions of genes related to the inflammation (IL-1ß and TNF-α) and as well as significantly up-regulated the expression of genes HSP70 and SOD. Additionally, the mRNA levels of MT gene can be speculated that the heavy metal cadmium may accumulated in the body over time. And the concentration of heavy metals will also affect their accumulation in the body. Our study elucidated the nano-microplastics and Cd will increase the impact on environmental and organisms that the nano-microplastics contribute to the bioaccumulation of metals, which served as a new support for study the interaction between environmental contaminants.

Effects of partial replacement of dietary flour meal with seaweed polysaccharides on the resistance to ammonia stress in the intestine of hybrid snakehead (Channa maculatus ♀ × Channa argus ♂).

The purpose of this study was to evaluate the effects of partial replacement of dietary flour meal with seaweed polysaccharides on survival rate, histology, intestinal oxidative stress levels, and expression of immune-related genes in hybrid snakeheads under acute ammonia stress. Four experimental diets were set: (C) basal diet with 0% of seaweed polysaccharides as the control group, (MR) basal diet with 10% of seaweed polysaccharides, (HR) basal diet with 15% of seaweed polysaccharides, (HF) basal diet with 10% of fish oil. After 60 days of feeding, fish fed with the diet of C group were sampled as the control group, and other fish were exposed to ammonia nitrogen for 48 h. Two concentrations of total ammonia nitrogen (TAN) were used in this study: 120 mg/L TAN (low concentration exposure group), and 1200 mg/L TAN (high concentration exposure group). After exposure to ammonia nitrogen for 48 h, fish were sampled. The results indicated that adding seaweed polysaccharides to the diet could improve the survival rate of hybrid snakeheads under high concentration of ammonia stress. Histopathological analysis demonstrated multiple abnormalities in gills and intestines after exposure to two concentrations of TAN. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and lactate dehydrogenase (LDH) were all increased in the MR group under two concentrations of TAN stress. The mRNA abundance of immune-related genes in fish intestinal tissues was significantly induced or inhibited. These results suggested that partial replacement of dietary flour meal with seaweed polysaccharides improved the ability of hybrid snakeheads to resist ammonia stress.

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.

Expression of genes related to antioxidation, immunity, and heat stress in Gambusia affinis exposed to the heavy metals Cu and Zn.

Water pollution is an increasingly serious problem. Here, Cu and Zn ions were used as stress factors, and G. affinis served as a test organism. Fluorescence quantitative PCR was used to detect changes in the expression of antioxidant genes (SOD, GST, CAT), heat stress genes (Hsp70, Hsp90, Hspd1, Hsc70), and immune system-related genes (IL-1ß, IL-8) in G. affinis exposed to Cu and Zn ions over time. To explore the toxic effects of Cu and Zn on G. affinis. The results showed that the 48 h LC50 concentrations of the heavy metals Cu and Zn to G. affinis were 0.17 mg/L and 44.67 mg/L, respectively. Within 48 h, with prolonged Cu exposure, the relative expression levels of the Hsp70, Hsp90, Hspd1, Hsc70, SOD, GST, and CAT genes in the gill tissue first showed a significant increase and then gradually decreased. Gene expression peaked between 9 and 36 h. The relative expression levels of SOD and GST genes in liver tissue showed a gradual decline. Within 48 h, with prolonged Zn exposure, the expression levels of SOD, CAT, and GST genes in G. affinis first increased and then fell before finally rising. The expression levels of IL-1ß and IL-8 mRNA showed varying degrees of upward trends, and the expression of IL-8 was the highest for all gill tissue. To sum up, Cu and Zn have strong toxic effects on G. affinis, which makes it possible to use G. affinis as indicator organisms for aquatic environmental pollution.

Gonadal Transcriptome Analysis and Sequence Characterization of Sex-Related Genes in Cranoglanis bouderius.

In China, the Cranoglanis bouderius is classified as a national class II-protected animal. The development of C. bouderius populations has been affected by a variety of factors over the past few decades, with severe declines occurring. Considering the likelihood of continued population declines of the C. bouderius in the future, it is critical to investigate the currently unknown characteristics of gonadal differentiation and sex-related genes for C. bouderius conservation. In this study, the Illumina sequencing platform was used to sequence the gonadal transcriptome of the C. bouderius to identify the pathways and genes related to gonadal development and analyze the expression differences in the gonads. A total of 12,002 DEGs were identified, with 7220 being significantly expressed in the ovary and 4782 being significantly expressed in the testis. According to the functional enrichment results, the cell cycle, RNA transport, apoptosis, Wnt signaling pathway, p53 signaling pathway, and prolactin signaling pathway play important roles in sex development in the C. bouderius. Furthermore, the sequence characterization and evolutionary analysis revealed that AMH, DAX1, NANOS1, and AR of the C. bouderius are highly conserved. Specifically, the qRT-PCR results from various tissues showed significant differences in AMH, DAX1, NANOS1, and AR expression levels in the gonads of both sexes of C. bouderius. These analyses indicated that AMH, DAX1, NANOS1, and AR may play important roles in the differentiation and development of C. bouderius gonads. To our best knowledge, this study is the first to analyze the C. bouderius gonadal transcriptome and identify the structures of sex-related genes, laying the foundation for future research.

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.

A dosage-effect assessment of acute toxicology tests of microplastic exposure in filter-feeding fish.

Abundant microplastics was found in aquatic ecosystem and aquatic organisms, which raised many concerns in public. Silver carp (Hypophthalmichthys molitrix), a species filter-feeding planktivorous fish, feed on particle between 4 and 85 µm in size, and the respiratory process works together with feeding mechanism when filtering plankton from water. The aim of this study was to assess the physiological response of silver carp exposed to 5 µm polystyrene microspheres during 48 h of exposure followed by 48 h of depuration through the gill histology, and oxidative stress biomarkers in intestine. The results revealed that microplastics can pass through the whole digestive tract of silver carp and be excreted by feces. Low microplastic concentration (80 µg/L) induced oxidative stress and up-regulation of TUB84 and HSP70 gene in intestine, and silver carp have ability to recover after the exposure to microplastic was removed. High microplastic concentration (800 µg/L) definitely cause significant damage to gills and intestines, in this situation, far beyond the possibility of fish own repair, and even when the threaten removed, silver carp can't recovery soon. Our studies assessed the dosage-effect relationship with physiological stress on silver carp when exposure to microplastics.

Characterization and spatial distribution of microplastics in two wild captured economic freshwater fish from north and west rivers of Guangdong province.

As a new type of pollutant, microplastics are of emerging widespread concern, while amount of research done in freshwater environments and organisms is litter compared to that in marine. Following this reality, the categories of MPs in two economic freshwater fish at 25 sites from 11 cities in the north and west rivers of Guangdong province were documented. Here, 76 individuals belong Oreochromis niloticus and Cirrhinus molitorella were investigated and microplastics were found in the GITs of 43.4% and gills of 25%. The average abundances of microplastics have significant difference between Oreochromis niloticus (0.015 items/ g) and Cirrhinus molitorella (0.031 items/g), while no difference by individual (~1.9 items). The plastics were dominated by white in color (61%), fragment in shape (67%), and lass than 1 mm in size (74%). The spatial distribution of microplastics revealed that there are significant differences between different cities in average abundances, and the highest average abundances of MPs were found in Zhanjiang city (4.25 items/individual) and Guangzhou city (0.044 items/g), respectively. Our results fully proved that the microplastics was widely ingested by wild fish species and suggested that the abundance and distribution of microplastics are positively related with the development of economy, tourism, industry, agriculture, and fishery.

Nanoplastics Induce More Serious Microbiota Dysbiosis and Inflammation in the Gut of Adult Zebrafish than Microplastics.

Microplastics (MPs) (< 5 mm) and nanoplastics (NPs) (< 100 nm) are emerging environmental pollutants and have been proved could cause a series of toxicity in aquatic organisms. In this study, the effects on gut microbiota of adult zebrafish exposed for 21 days to 10 µg/L and 1 mg/L of MPs (8 µm) and NPs (80 nm) were evaluated. We analyzed the intestinal microbial community of zebrafish using high throughput sequencing of the 16S rRNA gene V3-V4 region and also performed transcriptional profiling of the inflammation pathway related genes in the intestinal tissues. Our results showed that both spherical polystyrene MPs and NPs could induce microbiota dysbiosis in the gut of zebrafish. The flora diversity of gut microbiota significantly increased under a high concentration of NPs. At the phylum level, the abundance of Proteobacteria increased significantly and the abundance of Fusobacteria, Firmicutes and Verrucomicrobiota decreased significantly in the gut after 21-day exposure to 1 mg/L of both MPs and NPs. Furthermore, interestingly, the abundance of Actinobacteria decreased in the MPs treatment groups but increased in the NPs treatment groups. At the genus level, revealed that the relative abundance of Aeromonas significantly increased both in the MPs and NPs treatment groups. Moreover, it was observed that NPs increased mRNA levels of il8, il10, il1ß and tnfα in the gut, but not in MPs exposure group, indicating that the NPs may have a more serious effect on the gut of zebrafish than MPs to induce microbiota dysbiosis and inflammation in the gut.

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.

Immune-related genes expression analysis of Western mosquitofish (Gambusia affinis) challenged with Aeromonas hydrophila.

The great Gambusia affinis (G. affinis) is considered as an important animal model to study the endocrine disruption, ecological behavior, and environmental pollutant. The present study aims to build a new promising infection model with Aeromonas hydrophila (A. hydrophila) in aquaculture. The mRNA expression of Rac1, MyD88, IRAK4, TAK1, IKKß, and IL-1ß in G. affinis were significance higher (P < 0.05) in the liver of G. affinis than that of brain and intestine. And the PI3K mRNA expression level was significant lower (P < 0.05) in the intestine than that of brain and liver. The mRNA levels of AKT3 were significant higher (P < 0.05) in the brain than that of liver and intestine. And then the brain, liver, and intestine were collected at different time points (0 h, 3 h, 9 h, 18 h, 24 h, 48 h) after post injection of LD50 of A. hydrophila. The 0.85% NaCl was used as a negative control for the LD50 of A. hydrophila. The RT-PCR results showed that mRNA expressions of TLR2/4 pathway downstream genes MyD88, IRAK4, TAK1, Rac1, IKKß, and IL-1ß were firstly significantly up-regulated (P < 0.05) and were then backed to the 0 h group levels in three tissues. In contrast, mRNA expressions of TLR2/4 pathway downstream genes PI3K and AKT3 were firstly significantly decreased (P < 0.05) and were then increased to the 0 h group levels in brain and intestine. In summary, the results indicated that A. hydrophila could cause inflammatory reaction in intestinal and brain. In addition, the liver showed a provocative reaction when infected with A. hydrophila.

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.

Cytochrome P450 1A mRNA in the Gambusia affinis and Response to Several PAHs.

Cytochrome P4501A (CYP1A) has been used as a specific biomarker for monitoring water contamination such as PAHs, PCBs and dioxins. In the present study, the cyp1a gene of Gambusia affinis was cloned and sequenced and their expressions under PAHs exposure were characterized. The newly identified cyp1a encodes a protein with 521 amino acids that shared 96-80% identity with other Cyprinodontiformes fishes. RT-PCR analysis revealed that the basal mRNA level of cyp1a was highly expressed in liver and intestine. The expression level of cyp1a was significantly induced by exposure to 100 µg/L 3, 4-Benzopyrene (BaP) for 5 days in the muscle, testis, brain, liver and intestine of adult male fish. Except in the testis, the induced mRNA level of cyp1a ultimately decreased after prolonging the exposure time to 25 days. As for testis, the induced mRNA level of cyp1a was maintained at a high level during the entire exposure time under 100 µg/L BaP exposure. Furthermore, the expression of cyp1a increased with exposure time under a relatively low exposure concentrations 1 µg/L. Regarding the effects of other PAHs, D(a,h)A, BbF, and BaA showed a statistically significant effect of induction on mRNA level of cyp1a in the muscle, testis, brain, liver and intestine.

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.

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 dietary Gelsemium elegans alkaloids on growth performance, immune responses and disease resistance of Megalobrama amblycephala.

The present study aim to investigate the effects of dietary Gelsemium elegans alkaloids supplementation in Megalobrama amblycephala. A basal diet supplemented with 0, 5, 10, 20 and 40 mg/kg G. elegans alkaloids were fed to M. amblycephala for 12 weeks. The study indicated that dietary 20 mg/kg and 40 mg/kg G. elegans alkaloids supplementation could significantly improve final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) (P < 0.05). The 20 mg/kg and 40 mg/kg G. elegans alkaloids groups showed significantly higher whole body and muscle crude protein and crude lipid contents compared to the control group (P < 0.05). The amino acid contents in muscle were also significantly increased in 20 mg/kg and 40 mg/kg groups (P < 0.05). Dietary 40 mg/kg G. elegans alkaloids had a significant effect on the contents of LDH, AST, ALT, ALP, TG, TC, LDL-C, HDL-C, ALB and TP in M. amblycephala (P < 0.05). Fish fed 20 mg/kg and 40 mg/kg dietary G. elegans alkaloids showed significant increase in complement 3, complement 4 and immunoglobulin M contents. The liver antioxidant enzymes (SOD, CAT and T-AOC) and MDA content significantly increased at 20 mg/kg and 40 mg/kg G. elegans alkaloids supplement (P < 0.05). The mRNA levels of immune-related genes IL-1ß, IL8, TNF-α and IFN-α were significantly up-regulated, whereas TGF-ß and IL10 genes were significantly down-regulated in the liver, spleen and head kidney of fish fed dietary supplementation with 20 mg/kg and 40 mg/kg G. elegans alkaloids. After challenge with Aeromonas hydrophila, significant higher survival rate was observed at 20 mg/kg and 40 mg/kg G. elegans alkaloids supplement (P < 0.05). Therefore, these results indicated that M. amblycephala fed a diet supplemented with 20 mg/kg and 40 mg/kg G. elegans alkaloids could significantly promote its growth performance, lipids and amino acids deposition, immune ability and resistance to Aeromonas hydrophila.

Deletion of mstna and mstnb impairs the immune system and affects growth performance in zebrafish.

Myostatin (Mstn) is a negative regulator of muscle development in vertebrates. Although its function in muscle growth has been well studied in mammals and fish, it remains unclear whether or how mstn functions in the immune system. In this study, mstna-/- and mstnb-/- homozygous zebrafish were firstly generated using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). Deletion of mstnb but not mstna enhanced growth performance. Although survival rates under normal conditions were slightly decreased in both strains, mortality after dexamethasone-induced stress was increased by ∼30%. Furthermore, transcriptional levels of several critical immune-related genes were decreased, and the ability to withstand exposure to pathogenic E. tarda was decreased, compared with that of controls. In mstnb-/- but not mstna-/- zebrafish, expression of NF-κB subunits and several pro-inflammatory cytokines failed to respond to E. tarda exposure except nfkb1, c-rel and tnfα. Taken together, these results indicate that mstnb but not mstna plays a key role in zebrafish muscle growth. While each paralogue contributes to the response to bacterial insult, mstnb affects the immune system through activation of the NF-κB pathway, and mstna is likely to act upstream of NF-κB at some as yet unidentified target.