Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Comparative analysis of two new zebrafish models: The cyp1a low-expression line and cyp1a knockout line under PAHs exposure.

Cytochrome P450 1 (CYP1) is an important enzyme family involved in the metabolism of pollutants, and used as a biomarker to monitor environmental pollution. In this study, a fluorescence-labeled cyp1a zebrafish line, named as KI (cyp1a+/+-T2A-mCherry) (KICM), was originally constructed to monitor dioxin-like compounds in the environment. However, the cyp1a gene expression in the KICM line was inhibited by the fluorescence labeling, thus leading to a significantly increased sensitivity of KICM zebrafish line to PAHs. Then, a cyp1a knockout zebrafish line, named KOC, were constructed for comparative analysis with the cyp1a low-expression line. Interestingly, knockout of the cyp1a gene did not increase the sensitivity of zebrafish to PAHs as significantly as the cyp1a low-expression line. So, the expression levels of related genes in the aryl hydrocarbon receptor pathway were analyzed and the results showed that the expression level of cyp1b in KOC group was significantly higher than that of wild type and KICM under the same PAH exposure. This indicated that the effect of losing cyp1a was compensated by inducing expression of cyp1b. In conclusion, two new zebrafish models including cyp1a low-expression line and cyp1a knockout line were constructed in this study, which may provide a convenient model for subsequent studies on the toxicity mechanism of PAHs and the role of cyp1a in detoxification.

Species-specific effect of microplastics on fish embryos and observation of toxicity kinetics in larvae.

Microplastics will definitely increase the potential health risks to humans through food chain, especially by commercial fishes. Here, we studied species-specific effect of microplastics on fish embryos and observed uptake, accumulation and elimination of microplastics in larvae. We chose three commercial fish species with different feeding types as our research objects. The results we found demonstrated that microplastics abundance in larvae was related with feeding type. At the same exposure concentration, the ingestion of microplastics in carnivores was lower than that in filter feeders and omnivores. In addition, omnivores were less able to remove microplastics than filter feeders. To the best of our knowledge, this is the first study to compared the differences of microplastics ingested in fishes with feeding types under laboratory conditions, and we believe that the findings will be valid evidence to explain species-specific effect of microplastics on fishes.

Insights Into the Albinism Mechanism for Two Distinct Color Morphs of Northern Snakehead, Channa argus Through Histological and Transcriptome Analyses.

The great northern snakehead (Channa argus) is one of the most important economic and conservational fish in China. In this study, the melanocytes in the skin of two distinct color morphs C. argus were investigated and compared through employment of the microscopic analysis, hematoxylin and eosin (H&E) and Masson Fontana staining. Our results demonstrated the uneven distribution of melanocytes with extremely low density and most of them were in the state of aging or death. Meanwhile, there was no obvious pigment layer and melanocytes distribution pattern found in the albino-type (AT), while the melanocytes were evenly distributed with abundance in the bicolor-type (BT). The transcriptome analysis through Illumina HiSeq sequencing showed that a total of 34.93 Gb Clean Data was obtained, and Q30 base percentage reached 92.66%. The BT and AT northern snakeheads transcriptome data included a total of 56,039,701 and 60,410,063 clean reads (n = 3), respectively. In gene expression analyses, the sample correlation coefficients (r) were ranged between 0.92 and 1.00; the contribution of PC1 and PC2 were 50.25 and 13.73% by using PCA cluster analysis, the total number of DEGs were 1024 (559 up-regulated and 465 down-regulated), and the number of annotated DEGs was 767 (COG 172, KEGG 262, GO 288, SwissProt 548, Pfam 579 and NR 765). Additionally, 46,363 ± 873 and 44,947 ± 392 single nucleotide polymorphisms (SNPs) were compiled via genetic structure analysis, respectively. Ten key pigment-related genes were screened using qRT-PCR. And all of them revealed extremely higher expression levels in the skin of BT than those of AT. This is the first study to analyze the mechanism of albino characteristics of Channa via histology and transcriptomics, and also provide the oretical and practical support for the protection and development of germplasm resources for C. argus.

Effects of compound antimicrobial peptides on the growth performance, antioxidant and immune responses and disease resistance of grass carp (Ctenopharyngodon idellus).

In this study, the compound antimicrobial peptides was added to the basic diet to probe its effects on grass carp for 8 weeks. There were 6 groups in our study, including M0 (0 mg/kg) and 5 additional groups: M1 (100 mg/kg), M2 (200 mg/kg), M3 (400 mg/kg), M4 (800 mg/kg) and M5 (1600 mg/kg). The results are as follows: (1) The grass carp's FBW, WGR and SGR in M4 were significantly increased than M0 (p < 0.05). (2) In the hepatopancreas, the amylase activity of M2, M3 and M5 were significant up-regulation than other groups (p < 0.05). (3) The activity of T-AOC in the M3 and M4 was significantly higher compared to the other groups in grass carp hepatopancreas, while MDA was significantly reduced compared to the control group (p < 0.05). The activity of SOD in M5 was significantly increased than the other groups (p < 0.05). (4) The expression of MnSOD (except head kidney), IL8 and TNF-α and IL-1ß (except hepatopancreas) were significantly increased (p < 0.05), while TGF-ß and were significantly reduced in the hepatopancreas, spleen and head kidney at M3 (p < 0.05), and IL10 were significantly decreased in the hepatopancreas at M3 (p < 0.05). In addition, expression of IL8 and TNF-α were significant down-regulation, whereas TGF-ß (expect head kidney) were significant up-regulation in hepatopancreas, spleen and head kidney in M3 after challenge with A. hydrophila. The expression of IL-1ß in spleen and head kidney were also down-regulated, whereas IL10 were significantly up-regulated in the hepatopancreas at M3 after challenge with A. hydrophila (p < 0.05). The results of this study showed that grass carp fed a diet supplemented compound antimicrobial peptides was added with 400-800 mg/kg, which could improve the growth performance, antioxidant and digestive capabilities, and could also enhance the expression of immune-related genes and control to A. hydrophila.

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.

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.

Transcriptomic changes in western mosquitofish (Gambusia affinis) liver following benzo[a]pyrene exposure.

Widely distributed western mosquitofish (Gambusia affinis) has been used as a new model species for hazard assessment of environmental stressors such as polycyclic aromatic hydrocarbons (PAHs). However, most of the PAH studies using G. affinis rely on targeted biomarker-based analysis, and thus may not adequately address the complexity of the toxic mechanisms of the stressors. In the present study, the whole transcriptional sequencing of G. affinis liver after exposure to a PAH model, benzo[a]pyrene (BaP) (100 µg/L), for 20 days was performed by using the HiSeq XTen sequencers. In total, 58,156,233 and 51,825,467 clean nucleotide reads were obtained in the control and BaP-exposed libraries, respectively, with average N50 lengths of 1419 bp. In addition, after G. affinis was exposed for 20 days, 169 genes were upregulated, and 176 genes were downregulated in liver. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied to all the genes to determine the genes' biological functions and processes. The results clearly showed that the differentially expressed genes were mainly related to immune pathways and metabolic correlation pathways. Interestingly, almost all the pathways related with the immunity were upregulated, while the metabolism pathways were downregulated. Lastly, quantitative real-time PCR (qRT-PCR) was performed to measure expressional levels of twelve genes confirmed through the DGE analysis. These results demonstrate that BaP damages immunity and enhances the consumption of all available energy storage to activate mechanisms of the detoxification in G. affinis. Up until now, the present study is the first time that a whole transcriptome sequencing analysis in the liver of G. affinis exposed to BaP has been reported.

Integrative microRNA and mRNA analysis reveals regulation of ER stress in the Pacific white shrimp Litopenaeus vannamei under acute cold stress.

Litopenaeus vannamei (L. vannamei) is one of the most important aquaculture shrimps in the world and its survival, growth, and distribution was seriously challenged by cold stress. To investigate the response of L. vannamei under acute cold stress, mRNA and microRNA (miRNA) profiles of the hepatopancreas which was collected from the control group (28 °C) and the treatment group (13 °C) were analyzed. The results showed that 1266 differentially expressed genes (DEGs) (502 up- and 764 down-regulated) and 60 differentially expressed miRNAs (34 up- and 26 down-regulated) were identified under acute cold stress respectively. According to the functional annotation, catalytic activity was the most significant term in the GO category of molecular function, and protein processing in endoplasmic reticulum (ER) was the most enriched KEGG pathway for mRNAs profile. For miRNAs profile, functional annotation of the target genes of the differentially expressed miRNAs indicated that oxidoreductase activity was the most significant term in the GO category of molecular function, and glucagon signaling pathway was the most enriched KEGG pathway. The results revealed that ER involved in the acute cold stress response and acute cold stress may induce the unfolded protein response of L. vannamei.

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.

Induction of oxidative stress, apoptosis and DNA damage by koumine in Tetrahymena thermophila.

Koumine is a component of the Chinese medicinal herb Gelsemium elegans and is toxic to vertebrates. We used the ciliate Tetrahymena thermophila as a model to evaluate the toxic effects of this indole alkaloid in eukaryotic microorganisms. Koumine inhibited T. thermophila growth and viability in a dose-dependent manner. Moreover, this drug produced oxidative stress in T. thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine levels (p < 0.05). Koumine also caused significant levels of apoptosis (p < 0.05) and induced DNA damage in a dose-dependent manner. Mitophagic vacuoles were present in cells indicating induction of autophagy by this drug. Expression of ATG7, MTT2/4, CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine. This study represents a preliminary toxicological evaluation of koumine in the single celled eukaryote T. thermophila.

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.

Effects of gelsemine on oxidative stress and DNA damage responses of Tetrahymena thermophila.

Gelsemine is an important toxic substance extracted from Gelsemium elegans, which has a lot of biological functions in cells and organisms, but its toxicity has been rarely reported in Tetrahymena thermophila. In this study, we used the protozoan T. thermophila as an experimental model to investigate the potential toxicity-induced mechanism of gelsemine in the unicellular eukaryote. Our results clearly showed gelsemine inhibited T. thermophila growth in a dose-dependent manner. This exposure also resulted in oxidative stress on T. thermophila cells and antioxidant enzyme levels were significantly altered at high gelsemine levels (p < 0.05). Gelsemine produced a slight apoptotic effect at the highest (0.8 mg/mL) gelsemine level used here (p < 0.05). Furthermore, the toxin-induced DNA damage in a dose-dependent manner. The ultrastructural analysis also revealed mitophagic vacuoles at 0.4 and 0.8 mg/mL levels of gelsemine exposure. Moreover, expressions of oxidative stress-related and MAP kinase genes were significantly changed after exposure to 0.8 mg/mL level of gelsemine (p < 0.05). Altogether, our results clearly show that gelsemine from G. elegans can inhibit the growth via inducing oxidative stress and DNA damage in T. thermophila cells.

Generation of all-male-like sterile zebrafish by eliminating primordial germ cells at early development.

Production of all-male and sterile fish may not only substantially improve yield but also be crucial for the application of genome modified species in aquaculture. Previously, it was reported that the fish lacking primordial germ cells (PGCs) becomes infertile, and nitroreductase, an enzyme converting non-toxic metronidazole (MTZ) into toxic metabolites, induces targeted toxicity to kill the cells expressing it. In this study, we generated a transgenic zebrafish line of Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) in which the NfsB nitroreductase is solely expressed in PGCs. Treating the embryos derived from the female transgenic zebrafish with MTZ from 0 through 2 dpf (days post fertilization), we found that the germ cells were completely eliminated in the ones older than 2.5 dpf. At 20 dpf, the MTZ-treated juvenile had no germ cells in their gonads. At 100 dpf, the MTZ-treated adult exhibited male-like morphology and showed normal mating behaviors although they had no germ cells but only supporting cells in their gonads. Taken together, our results demonstrated that conditional elimination of PGCs during early development make the zebrafish male-like and infertile. It may provide an alternative strategy to make sterile and all-male farmed fish that is good for increasing aquaculture yield and preventing the genome modified species from potential ecological risks.