Systematic Identification of Long Noncoding RNAs during Three Key Organogenesis Stages in Zebrafish.

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish.

Adverse effects of fenpropathrin on the intestine of common carp (Cyprinus carpio L.) and the mechanism involved.

Fenpropathrin (FEN), a pyrethroid pesticide, is frequently detected in natural water bodies, unavoidable pose adverse effects to aquatic organisms. However, the harmful effects and potential mechanisms of FEN on aquatic species are poorly understood. In this study, common carp were treatment with FEN at 0.45 and 1.35 µg/L for 14 d, and the toxic effects and underlying mechanisms of FEN on the intestine of carp were revealed. RNA-seq results showed that FEN exposure cause a wide range of transcriptional alterations in the intestine and the differentially expressed genes were mainly enrichment in the pathways related to immune and metabolism. Specifically, FEN exposure induced pathological damage and altered submicroscopic structure of the intestine, elevated the levels of Bacteroides fragilis enterotoxin, altered the contents of claudin-1, occludin, and zonula occluden-1 (ZO-1), and causing injury to the intestinal barrier. In addition, inflammation-related index TNF-α in the serum and IL-6 in the intestinal tissues were generally increased after FEN exposure. Moreover, FEN exposure promoted an increase in reactive oxygen species (ROS), altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), upregulated the contents of malondialdehyde (MDA) in the intestines. The apoptosis-related parameter cytochrome c, caspase-9, and caspase-3 were significantly altered, indicating that inflammation reaction, oxidative stress, and apoptosis may be involved in the toxic mechanism of FEN on carp. Moreover, FEN treatment also altered the intestinal flora community significantly, which may affect the intestinal normal physiological function and thus affect the growth of fish. Overall, the present study help to clarify the intestinal reaction mechanisms after FEN treatment, and provide a basis for the risk assessment of FEN.

Fenpropathrin induces neurotoxic effects in common carp (Cyprinus carpio L.).

Fenpropathrin (FEN) is a synthetic pyrethroid that has been frequently detected in aquatic environments, yet the neurotoxic impacts and underlying mechanisms on nontarget organisms are lacking. In this experiment, common carp were exposed to 0.45 and 1.35 µg/L FEN for 14 d and exhibited abnormal locomotor behaviour. Biochemical and molecular analysis results indicated that FEN altered the contents of tight junction proteins (claudin-1, occludin, and ZO-1), disturbed Na+-K+-ATPase and AChE activities, caused abnormal expression of neurotransmitters (ACh, DA, GABA, 5-HT, and glutamate) and caused histological damage in the brain, suggesting that FEN may damage the blood-brain barrier and induce neurotoxicity in carp. Furthermore, FEN also promoted an increase in ROS, changed SOD and CAT activities, and generally upregulated the contents of MDA, 8-OHdG, and protein carbonyl in the brain, indicating that FEN can induce oxidative stress and cause damage to lipids, DNA, and proteins. Moreover, inflammation-related indicators (TNF-α, IL-1ß, IL-6, and IL-10), mitophagy-related genes (PINK1, parkin, ulk1, beclin1, LC3, p62, tfeb, and atg5), and apoptosis-related parameters (p53, bax, bcl-2, caspase-3, caspase-8, and caspase-9) were also significantly changed, suggesting that inflammation, mitophagy, and apoptosis may participate in FEN-induced neurotoxicity in carp. This study refines the understanding of the toxicity mechanism of FEN and thus provides data support for the risk assessment of FEN.

Environmental impacts of chlorpyrifos: Transgenerational toxic effects on aquatic organisms cannot be ignored.

Chlorpyrifos (CPF) has been extensively used in the world and frequently found in natural environments, might cause a range of environmental issues and pose a health risk to aquatic species. However, investigation of its toxic effects on offspring after parental exposure has been neglected, especially for aquatic organisms such as fish. In the current study, the effects of chronic CPF exposure (3 and 60 µg/L) on adult zebrafish (F0) was investigated to determine its influence on adult reproductive capacity and offspring (F1 and F2). The results showed the existence of CPF both in F0 ovaries and F1 embryos and larvae, indicating that CPF could be transferred directly from the F0 adult fish to F1 offspring. After 90 d exposure, we observed that F0 female fish showed increased proportion of perinucleolar oocyte in the ovaries, decreased proportion of mature oocyte, and decreased egg production, but not in F1 adult. The transcriptomic analysis revealed that the disruption of metabolism during oocyte maturation in the CPF treatment zebrafish might interfere with F0 oocytes development and quality and ultimately influence offspring survival. For the larvae, the parental CPF exposure distinctly inhibited heart rate at 72 and 120 hpf and increased the mortality of F1 but not F2 larvae. The changes of biochemical indicators confirmed a disturbance in the oxidative balance, induced inflammatory reaction and apoptosis in F1 larvae. Furthermore, the changing profiles of mRNA revealed by RNA-seq confirmed an increased susceptibility in F1 larvae and figured out potential disruptions of ROS metabolism, immune system, apoptosis, and metabolism pathways. Taken together, these results show that chronic CPF treatment can induce reproductive toxicity, and parental transfer of CPF occurs in fish, resulting in transgenerational alters in F1 generation survival and transcription that raising concerns on the ecological risk of CPF in the natural environment.

High-throughput transcriptome sequencing reveals the key stages of cardiovascular development in zebrafish embryos.

BACKGROUND: The cardiovascular developmental process is a tightly regulated network involving multiple genes. The current understanding of the molecular mechanism behind cardiovascular development is insufficient and requires further research. RESULTS: Transcriptome sequencing of three developmental stages in zebrafish embryos was performed and revealed three key cardiovascular developmental stages. Then, the differentially expressed genes (DEGs) involved in cardiovascular development were screened out. The three developmental stages were 18 (T1), 24 (T2), and 42 h post fertilization (hpf) (T3), and the three stages were confirmed by detecting differences in expression between cardiomyocyte and endothelial marker genes (cmlc2, fli1) using in situ hybridization, which represents the characteristics of cardiovascular development. Thousands of DEGs were identified using transcriptome analysis. Of them, 2605 DEGs were in T1-vs-T2, including 2003 up-regulated and 602 down-regulated genes, 6446 DEGs were in T1-vs-T3, consisting of 4608 up-regulated and 1838 down-regulated genes, and 3275 DEGs were in T2-vs-T3, including 2420 up-regulated and 855 down-regulated genes. There were 644 common DEGs and 167 common five-fold higher differentially expressed genes (HDEGs) identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Significant differences was observed in the levels of gene expression among different developmental stages in multiple GO terms and KEGG pathways, such as cell migration to the midline involved in heart development, cardiovascular system development, circulatory system process for biological processes of GO terms; and cardiac muscle contraction, adrenergic signaling in cardiomyocytes for KEGG pathways. These results demonstrated that these three stages were important period for the development of the cardiovascular system. Lastly, we used quantitative real-time PCR (qPCR) to validate the reliability of RNA-sequencing by selecting 21 DEGs. CONCLUSIONS: These results demonstrated that these three stages represented the important periods for cardiovascular system development of zebrafish and some candidate genes was obtained and provided a solid foundation for additional functional studies of the DEGs.

Inhibition Effect of Ionic Liquid [Hmim]Cl on Microcystis Growth and Toxin Production.

Ionic liquids (ILs) are known as "green solvents" and widely used in industrial applications. However, little research has been conducted on cyanobacteria. This study was conducted to investigate the toxicity of ionic liquids ([Hmim]Cl) on Microcystis aeruginosa PCC7806. The EC50 (72 h) of [Hmim]Cl on the growth of Microcystis aeruginosa PCC 7806 was 10.624 ± 0.221 mg L-1. The possible mechanism of toxicity of [Hmim]Cl against M. aeruginosa PCC 7806 was evaluated by measuring cell growth, photosynthetic pigment contents, chlorophyll fluorescence transients, cell ultrastructure, and transcription of the microcystin-producing gene (mcyB). The concentrations of chlorophyll a and carotenoids were significantly reduced in treated M. aeruginosa cultures. The results of chlorophyll fluorescence transients showed that [Hmim]Cl could destruct the electron-accepting side of the photosystem II of M. aeruginosa PCC 7806. Transmission electron microscopy demonstrated cell damage including changes in the structure of the cell wall and cell membrane, thylakoid destruction, and nucleoid disassembly. The transcription of the mcyB gene was also inhibited under [Hmim]Cl stress. In summary, this study provides new insights into the toxicity of [Hmim]Cl on cyanobactreia.

Neurotoxicity of Chronic Co-Exposure of Lead and Ionic Liquid in Common Carp: Synergistic or Antagonistic?

Previous studies have indicated that the harmful heavy metal lead (Pb) contamination in aquatic systems has caused intelligence development disorders and nervous system function abnormalities in juveniles due to the increased permeability of the blood-brain barrier. Ionic liquids (ILs) are considered "green" organic solvents that can replace traditional organic solvents. Studies have found the presence of ILs in soil and water due to chemical applications or unintentional leakage. Therefore, what would happen if Pb interacted with ILs in a body of water? Could ILs enable Pb to more easily cross the blood-brain barrier? Therefore, we examined the combined exposure of Pb and ILs in common carp at low concentration (18.3 mg L-1 of Pb(CH3COO)2•3 H2O and 11 mg L-1 of the IL 1-methyl-3-octylimidazolium chloride, 5% of their LC50) for 28 days in the present study. The result of a neurobehavioral assay showed that chronic exposure of lead at lower concentrations significantly altered fish movement and neurobehaviors, indicating that lead exposure caused neurotoxicity in the carp. Increases in the neurotransmitter dopamine levels and injuries in the fish brain accounted for neurobehavioral abnormalities induced by lead exposure. Moreover, we also found that lead could easily cross the blood-brain barrier and caused significant bioaccumulation in the brain. Particularly, our study indicated that the ionic liquid could not synergistically promote blood-brain barrier permeability and hence failed to increase the absorption of lead in the fish brain, suggesting that the combined exposure of lead and ILs was not a synergistic effect but antagonism to the neurotoxicity. The results of this study suggested that ILs could recede the Pb induced neurotoxicity in fish.

Effect of Acute Exposure to the Ionic Liquid 1-Methyl-3-octylimidazolium Chloride on the Embryonic Development and Larval Thyroid System of Zebrafish.

Previous studies have shown that ILs can induce toxicity in animals, plants, and cells. However, the effect of imidazolium-based ILs on the hypothalamus-pituitary-thyroid (HPT) axis of fish remains unknown. The present study aimed to evaluate the acute effect of [C8mim]Cl on the embryonic development and thyroid-controlled internal secretion system of zebrafish by determining the thyroid hormone level and the expression of HPT-related genes. The results obtained for embryonic developmental toxicity showed the survival rate, heart beats, and body length of fish had decreased 96 h after exposure to [C8mim]Cl, but the hatching rate had increased by the 48 h time point. The transcription levels of HTP-related genes showed that the genes dio3, tg, ttr, tsh, trhrα, trhrß, trhr2, and tpo were up-regulated, while the expression levels of dio1, trh, tshr, and nis were significantly suppressed. Furthermore, we found that exposure to [C8mim]Cl induced an alteration in the levels of thyroid hormones that increased the T3 but decreased the T4 content. In conclusion, our study indicated that acute exposure to [C8mim]Cl altered the expression of HTP-related genes and disturbed the thyroid hormone level, suggesting that the ionic liquid [C8mim]Cl might pose an aquatic environmental threat to fish.

Developmental toxicity of glyphosate on embryo-larval zebrafish (Danio rerio).

Glyphosate (GLY) induces developmental toxicity in fish, but research on the toxicity mechanism is limited. In this study, zebrafish embryos were exposed for 120 hpf to 0.7, 7, and 35 mg L-1 GLY. The results show that GLY treatment induced developmental toxicity in the fish, including premature hatching, reduced heartbeats, pericardial and yolk sac oedema, swim bladder deficiency, and shortened body length, which was possibly due to a significantly decreased triiodothyronine (T3)/thyroxine (T4) ratio and the abnormal expression patterns of hypothalamic-pituitary-thyroid (HPT) (crh, tshß, tr α, tr ß, and t tr ) and growth hormone/insulin-like growth factor (GH/IGF) axis-related genes (gh, ghrα, ghrß, igf1, igf1rα, and igf1rß) in larvae exposed to GLY. In addition, GLY exposure altered the levels of SOD and CAT, increased ROS, promoted malondialdehyde (MDA) content, and significantly altered the levels of endoplasmic reticulum (ER) stress signalling pathway factors (perk, eif2α, gadd34, atf4, ire1α, xbp1, atf6, hspa5, and chop), suggesting that GLY treatment induced oxidative injury and ER stress in the larvae. Further research showed that treatment with a higher concentration of GLY upregulated the levels of iNOS, IL-1ß, and TNF-α while inhibiting the expression of IL-10 and TGF-ß, suggesting that GLY causes an inflammatory reaction in the larvae. In addition, we also found that apoptosis was induced in the larvae, which was determined by acridine orange staining and abnormal expression of p53, caspase-3, -8, and -9. Taken together, our results demonstrate that GLY exposure altered the T3/T4 ratio, disturbed the expression patterns of HPT and GH/IGF axis-related genes, and induced oxidative and ER stress, inflammatory reactions, and apoptosis in the zebrafish larvae. This investigation contributes to improved understanding of the developmental toxicity mechanism of GLY in fish.

Effects of polyvinyl chloride microplastics on reproduction, oxidative stress and reproduction and detoxification-related genes in Daphnia magna.

The drastic increase of microplastics (MPs) in aquatic environment has become a serious threat to marine and freshwater ecosystems. However, little information is available regarding the potential detrimental effects of polyvinyl chloride microplastics (PVC-MPs) on aquatic organisms. This study investigated the changes of reproduction parameters, oxidative stress and the expression of reproduction and detoxification-related genes in Daphnia magna after exposed to 2 ± 1 and 50 ± 10 µm PVC-MPs. The results showed that chronic exposure to 2 ± 1 µm PVC-MPs prolonged days to the first brood, increased total number of broods per female and frequency of molting per adult, decreased offspring number at first brood and total number of offspring per female in D. magna. Moreover, 2 ± 1 µm PVC-MPs also disturbed the activities of SOD and CAT, increased GSH and MDA levels. The expression of Vtg, SOD, CAT, CYP314 and CYP360A8 genes also exhibited different response patterns depending on exposure time. Furthermore, 50 ± 10 µm PVC-MPs decreased offspring at first brood and Vtg mRNA level, increased the transcription levels and activities of SOD and CAT. These results suggest that the presence of PVC-MPs in aquatic environment may cause reproduction toxicity by disrupting the reproduction and detoxification-related genes expression and inducing oxidative stress in D. magna.

Negative impacts of microcystin-LR and glyphosate on zebrafish intestine: Linked with gut microbiota and microRNAs?

Microcystin-LR (MC-LR) and glyphosate (GLY) have been classified as a Group 2B and Group 2A carcinogens for humans, respectively, and frequently found in aquatic ecosystems. However, data on the potential hazard of MC-LR and GLY exposure to the fish gut are relatively scarce. In the current study, a subacute toxicity test of zebrafish exposed to MC-LR (35 µg L-1) and GLY (3.5 mg L-1), either alone or in combination was performed for 21 d. The results showed that MC-LR or/and GLY treatment reduced the mRNA levels of tight junction genes (claudin-5, occludin, and zonula occludens-1) and altered the levels of diamine oxidase and D-lactic, indicating increased intestinal permeability in zebrafish. Furthermore, MC-LR and/or GLY treatment remarkably increased the levels of intestinal IL-1ß and IL-8 but decreased the levels of IL-10 and TGF-ß, indicating that MC-LR and/or GLY exposure induced an inflammatory response in the fish gut. MC-LR and/or GLY exposure also activated superoxide dismutase and catalase, generally upregulated the levels of p53, bax, bcl-2, caspase-3, and caspase-9, downregulated the levels of caspase-8 and caused notable histological injury in the fish gut. Moreover, MC-LR and/or GLY exposure also significantly altered the microbial community in the zebrafish gut and the expression of miRNAs (miR-146a, miR-155, miR-16, miR-21, and miR-223). Chronic exposure to MC-LR and/or GLY can induce intestinal damage in zebrafish, and this study is the first to demonstrate an altered gut microbiome and miRNAs in the zebrafish gut after MC-LR and GLY exposure.

Expression analysis of Hsp90α and cytokines in zebrafish caudal fin regeneration.

Zebrafish (Danio rerio) is an ideal model organism for exploring the ability and mechanism of tissue regeneration in the vertebrate. However, the specific cellular and molecular mechanism of caudal fin regeneration in zebrafish remains largely unclear. Therefore, we first confirmed the crucial period of fin regeneration in adult zebrafish by morphological and histological analysis. Then we performed RNA-Seq analysis of the caudal fin regeneration at three key stages, which provided some clues for exploring the mechanism of caudal fin regeneration. Moreover, we also determined the expressions of inflammatory cytokines IL-1ß, IL-6, IL-8, IL-10, TGF-ß, and the immune-related pathway JAK2α and STAT1b in the caudal fin of zebrafish following fin amputation by quantitative real time PCR (qPCR). Particularly, Hsp90α expression at mRNA and protein level determined by qPCR and Western blotting, respectively, and whole-mount in situ hybridization of Hsp90α were also performed in this study. The results showed that inflammatory cytokines were mainly expressed in the early period of caudal fin regeneration (1-3 days post amputation, dpa), indicating that fish immune system was involved in the fin regeneration. Furthermore, the high expression of Hsp90α in the vicinity of blastema and blood vessels of the regenerating fin suggests that Hsp90α may play a role in the initiation and promotion of caudal fin regeneration. Overall, our results provide a framework for further understanding the cellular and molecular mechanism in caudal fin regeneration.

Antimicrobial and anticancer activities of Scenedesmus obliquus metabolites.

This study was conducted to evaluate the antimicrobial activities and cytotoxicity of both crude extracts of Scenedesmus obliquus and their fractions. In vitro cytotoxicity assay against human hepatocellular carcinoma (HepG2), colon cancer (HCT116) and breast cancer (MCF7) cell line was monitored. The highest inhibition was observed using diethyl ether crude extract (DEE) recording between 12.5 and 19.5 mm inhibition zone against all tested bacteria and between 8.7 and 18.3 mm against tested fungi. The highest anticancer effect of DEE was observed at IC50 against HCT116 and HepG2 cell lines using just 24.6 and 42.8 µg ml-1, respectively. While, high concentration, 93.8 µg ml-1, was required to exhibit its effect against MCF7. Column chromatography technique was used to separate DEE crude extract to its main components using 7 different mobile phases. Fractions F1 and F7 were the highest fractions that had antimicrobial activity against tested bacteria and fungi. High IC50 > 80 µg ml-1 were required to exhibit anticancer activity at IC50 against the tested cancer cell lines. The main compounds responsible for the bioactivity were identified using GC-MS, nonadecane and butylated hydroxytoluene in F1 and 9-octadecadienoic acid and quercetin 7,3',4'-trimethoxy in F7 were identified. The current study highlights the potential use of S. Obliquus extract and their fractions as a source of antimicrobial and anticancer compounds.

Bio-evaluation of crustacean and fungal nano-chitosan for applying as food ingredient.

Chitosan, bio-polyaminosacharide, is derived from chitin. Two sources (shrimp wastes and fungus biomass) were used to produce chitosan. And then the chitosan was produced in the nano-form followed by characterization by transmission electron microscopy. The images obtained clearly showed that the size of nano-chitosan ranged between 7 and 13 and 3-6 nm with spherical shape for shrimp and fungal sources, respectively. The antimicrobial activities of the tested concentrations of chitosan and nano-chitosan were examined and found to have high activity against the tested pathogens. The evaluation of the toxicity of the tested concentrations of the produced chitosan and its nano-size were performed using brine shrimp and rat bioassay. Toxicity examination of chitosan and their nano derivatives is an essential procedure to assess the possibility of using these concentrations as food ingredient. Nine groups of rats were treated with either chitosan or nano-chitosan of both sources at 100 and 200 mg kg-1 bw. Adding chitosan in the diet of all groups showed no significant changes in both the blood biochemical and oxidative stress parameters when compared with control group. The histopathology of liver, kidney and stomach confirmed the results of the previous parameters. No signs of inflammation, fibrosis or cirrhosis were found in examined organs. It is concluded that chitosan and nano-chitosan of shrimp and Rhizopus stolonifer had high antimicrobial activity and are not toxic in the same time and it can be used as food ingredients.

Mycotoxigenic fungi in peanuts from different geographic regions of Egypt.

To understand the importance of mycotoxigenic fungi in Egyptian peanuts, samples from five regions (Alexandria, El-Beheira, El-Sharqiya, El-Daqahelaya in northern Egypt and Asyut, southern Egypt) in two seasons (2007, 2008) were collected. Aspergillus was consistently the most frequent genus in seeds and in-shell peanuts and was the dominant mycotoxigenic component of the mycobiota. There was no direct correlation between the moisture content of the samples and the fungal populations on peanut seeds tested from different regions. The most common species were from Aspergillus section Flavi (4.7-78.3%), Aspergillus section Nigri (9.4-52.6%) and Aspergillus section Circumdati (5.1-30.9%). In the in-shell peanut samples, the lowest populations were recorded in El-Beheira and Asyut (3.7-4.0 log10 CFU g(-1)) and the highest in Alexandria and Elsharqiya (4.1-6.0 log10 CFU g(-1)). Aspergillus section Flavi and section Nigri were the most dominant, and Aspergillus section Circumdati were only found in samples in 2008. Both qualitative (coconut cream agar) and quantitative analyses (HPLC) were used to analyse the potential mycotoxin production by strains isolated from peanuts. Of a total of 88 Aspergillus section Flavi strains examined, 95% were A. flavus based on production of aflatoxin B1 on yeast extract sucrose (YES) medium and confirmation using molecular analyses. Of 64 Aspergillus section Circumdati strains only 28% produced ochratoxin A (OTA), and were identified as A. westerdijkiae. No Aspergillus section Nigri strains produced OTA, and they were identified as A. niger (uniseriate). The presence of these toxigenic fungi indicates that there is a potential risk of mycotoxin contamination in Egyptian peanuts and suggests that problems can arise from contamination with both aflatoxins and perhaps also OTA.