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Microcystin-LR (MC-LR) is regarded as one of the most toxic microcystins (MCs) isoforms. Microcystins could cause multiple organs dysfunction, and more attention has been drawn to the toxic effects on the gastrointestinal disorder. By using ex vivo everted gut sac model in 6 fish (Carassius auratus, Megalobrama amblycephala, Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idellus and Cyprinus carpio) and determining the accumulation of MC-LR in zebrafish intestine, we found a dose-dependent manner in the absorption and accumulation of MC-LR. Until now, little studies have been reported concerning the gut microbiota composition caused by different MC-LR exposure. The present study is the first time characterized the phylogenetic composition and taxonomic of the bacterial communities growth in the intestines of zebrafish treated with MC-LR using 16S rRNA pyrosequencing. After 30 days of treatment with 0, 1, 5 or 20 μg/L MC-LR, the alpha and beta diversity did not generate significant differences, indicating the existence of a core microbiota. However, db-RDA analysis showed that treatment with 20 μg/L MC-LR changed the characteristics of high abundances microbiota. The expression of Oatp2b1, stress related enzyme activities in gut and their associations with gut microbiota were also determined. The identified phylotypes including Actinobacteria, Lactobacillus and some opportunistic pathogens highlight the increasing risks of pathogen invasion and recovery tendency via potential probiotics resistance in zebrafish exposed to MC-LR.

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.

Cyanobacterial blooms and their secondary metabolites, microcystins (MCs), are not only toxic to aquatic organisms, but also to humans. MCs exert reproductive toxicity in female fish by affecting the oocyte development. However, the mechanism behind MC-LR interference in oocyte development remains largely unknown. In our study, adult female zebrafish were exposed to MC-LR (0, 1, 5, 20 μg/L) for 30 d. After exposure to MC-LR for 30 d, fertilized eggs from the treated females and healthy males were collected and cultured in water without MC-LR. Histomorphological observations showed pathological damage in the ovary after MC-LR exposure, which was mainly characterized by enlarged intercellular spaces, detachment of follicular cells from oocytes, and vacuolation of parenchymal tissues. The 20 μg/L MC-LR treatment caused a remarkable increase in the rate of the zebrafish oocytes germinal vesicle breakdown (GVBD) and a significant decrease in the levels of cyclic adenosine monophosphate (cAMP) and vitellogenin (VTG). In addition, the phosphorylation levels of the extracellular signal-regulated kinases (ERK) were elevated in ovaries from zebrafish exposed to 5 and 20 μg/L MC-LR, and cyclinB phosphorylation levels were also upregulated notably in the 20 μg/L MC-LR group. However, MC-LR exposure did not cause any change in the levels of cAMP-dependent protein kinase (PKA) protein and cdc2 phosphorylation in all the treatments. All the doses of MC-LR reduced the number of eggs, prematurely hatched the fertilized eggs and increased the abnormal rate of offspring generation. In summary, the present study demonstrates that MC-LR promotes oocyte maturation by activating the ERK1/2 and MPF signaling pathways, and cAMP is involved in this process.

The existence of microcystins (MCs), the secondary metabolite of cyanobacteria, has become a growing public health concern. Previous researches have proved that MCs can trigger endoplasmic reticulum stress (ERS), but the underlying mechanisms remain unclear. In the present study, adult female zebrafish were exposed to MC-LR (0, 1, 5 and 20 μg/L) for 30 d, and the offspring derived from the treated females and healthy males were cultured in water without MC-LR until 96 h post fertilization (hpf). Our data suggested that MC-LR causes a significant increase in the eif2s1a, atf4, and eif2ak3 transcription levels in the liver and ovary. The mRNA levels of atf4, atf6, bcl-2, hspa5, eif2s1a and eif2ak3 upregulated notably in the offspring. JNK phosphorylation level and cleaved-caspase3 protein expression elevated obviously in the liver and ovary, but had no remarkable change in the offspring. Furthermore, TUNEL results showed that MC-LR significantly induced apoptosis in the liver and ovary, while acridine orange (AO) staining indicated that MC-LR did not cause abnormal apoptosis in offspring. Concisely, the present study indicated that MC-LR leads to apoptosis through different ERS pathways in the liver, ovary and offspring, and also provides a new perspective for understanding the apoptosis caused by MC-LR.

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 μg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 μg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 μg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 μg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 μM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 μg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.

Microcystins (MCs) released during cyanobacterial blooms exert varied toxicity on fish. Up to now, the reproductive toxicity of MCs on fish has rarely been reported. The present study investigated the reproductive toxicity of microcystin-LR (MC-LR) on male and female zebrafish (Danio rerio) by subchronic immersion in 1, 5, 20 μg/L for 30 d. After MC-LR exposure, the hatchability and the 17 beta-estradiol (E2) concentration in gonads significantly decreased in the 20 μg/L group. In the 5 and 20 μg/L groups, the whole body vitellogenin (VTG) levels significantly increased in females, while considerably decreased in males. The VTG1 transcriptional level significantly reduced in the liver of both female and male treated fish. Marked histological lesions were observed in the livers, ovaries and testes in MC-LR treated fish. Apoptotic rate in the ovaries significantly increased. Significant down-regulation of Bcl-2 transcriptional level was found in the gonads of all MC-LR treated fish, while marked up-regulation of Bax transcription level was determined in the 20 μg/L female treatment group, but a significant down-regulation in males. Although the transcriptional level of caspase-3 dropped in ovaries of the 5 and 20 μg/L treatment groups, the significant increase of caspase-3 activation levels in the ovaries and testes were detected. The present findings indicate that MC-LR exposure exerts diverse reproductive toxicity in zebrafish with females exhibiting more sensitivity than males. The present study also confirmed for the first time that MC-LR does not cause any estrogenic effects in adult zebrafish.

While microcystins (MCs) have been reported to exert reproductive toxicity on fish with a sex-dependent effect, the underlying mechanism has been rarely investigated. In the present study, zebrafish were exposed to 1, 5 and 20 μg/L MC-LR for 30 d. The gonad-somatic index declined in all treated males. 17β-estradiol (E2), testosterone (T), 11-keto testosterone (11-KT) and follicle-stimulating hormone (FSH) levels increased in serum from all treated females, while T, FSH and luteinizing hormone (LH) levels changed in all treated males. Histomorphological observation showed that MC-LR exposure evidently retarded oogenesis and spermatogenesis. Transcriptional changes of 22 genes of the hypothalamic-pituitary-gonad (HPG) axis exhibited sex-specific responses and the relationship between gene transcriptions and gametogenesis was evaluated by principle component analysis (PCA). Major contributors to PC1 (gnrh2, gnrhr3, ar, lhr, hmgra, hmgrb and cyp19a) were positively correlated with the number of post-vitellogenic oocytes, while PC1 (gnrh2, lhβ, erβ, fshr, cyp11a and 17βhsd) were positively correlated with the number of spermatozoa. The protein levels of 17βHSD and CYP19a were affected in both females and males. In conclusion, this study first investigated the sex-dependent effects of microcystins on fish reproduction and revealed some important molecular biomarkers related to gametogenesis in zebrafish suffered from MC-LR.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs), especially microcystin-LR (MC-LR), released during cyanobacterial blooms exert great toxicity on fish and even lead to massive death. The present study mainly investigated the pathological damage and immune response of spleen, gut and gill in zebrafish exposed to MC-LR. Fish were exposed to 0, 1, 5 and 20 μg/L of MC-LR for 30 d. In zebrafish exposed to 5 and 20 μg/L MC-LR, edematous mitochondria, deformation of the nucleus and compaction of chromatin were observed in lymphocyte of spleen; frayed gut villi, exfoliation of epithelial cells and widespread cell lyses were observed in intestines; hyperemia in gill lamellae, epithelial tissue edema and uplift and lamellar fusion were observed in gill. Varied changed gene expression was observed in spleen, intestine and gill of zebrafish. The transcriptional levels of IFN-1 and IL-8 in spleen significantly up-regulated in 20 μg/L group, and the transcription of IL-1β and TNFα in spleen increased in 1 μg/L MC-LR treated fish. In addition, the mRNA levels of IFN-1, IL-1β, IL-8, TGF-β and TNF-α dramatically increased in intestine and gill in all MC-LR treated groups. The present studies indicated that MC-LR exposure caused marked pathological damage, however, fish could adjust actively the expression of innate immune-related genes to resist the tissue damage. Our findings provided strong evidence of the recovery potential of fish exposed to microcystins.

Microcystins (MCs) are algal toxins produced intracellularly within the cyanobacteria cells. MCs exposure exerts great harm to the reproductive system of fish and deteriorates the quality of eggs and sperms, and has further adverse effects on early developmental stages of fish. Whether the MC toxicity can be parentally transmitted to offspring, even though the embryos and larvae are free of MC exposure? In the present study, adult zebrafish were continuously exposed to MC-LR (with dose of 1, 5 and 20 μg/L) for 30 days. After MC-LR exposure, fertilized eggs were collected and the following F1 generation was reared in water containing no MC-LR until 60 days post fertilization (dpf). In F1 offspring, both body weight and body length were evidently dropped. Some growth and immune related genes were detected using the real-time PCR. The transcriptional levels of these genes significantly decreased in F1 offspring of zebrafish whose parents were treated with 5 and 20 μg/L MC-LR. The activities of some antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) significantly dropped in 5 and 20 μg/L MC-LR groups, and the malondialdehyde (MDA) levels markedly increased in all the three treatment groups. Furthermore, distinct pathological changes in liver were observed in F1 zebrafish. Our findings show that the MC-LR exposure to parental zebrafish results in liver damage and evidently influences the growth and immune function in F1 offspring. We consider this damage as a parental transmission effect of microcystin toxicity. Further mechanism studies are necessary to elucidate this transmission effect.