Involvement of reactive oxygen species (ROS) in the hepatopancreatic cytotoxicity, oxidative stress, and apoptosis induced by microcystin-LR in Eriocheir sinensis.

There is limited knowledge about the toxicity of Microcystin-LR (MC-LR) in crustaceans, despite its high toxicity to aquatic organisms. This research aimed to explore the effects of MC-LR on cytotoxicity, oxidative stress, and apoptosis in the hepatopancreas of Eriocheir sinensis, as well as elucidate the involvement of reactive oxygen species (ROS) and potential mechanisms of toxicity. In vivo and in vitro exposures of crabs to MC-LR and N-acetylcysteine (NAC) were performed, followed by assessments of cell morphology, viability, tissue pathology, biochemical indicators, gene expression, and hepatopancreatic transcriptome. Results revealed that MC-LR facilitated the entry of the MC-LR transporter oatp3a into hepatopancreatic cells, leading to upregulated expression of phase I detoxification enzyme genes (cyp4c, cyp2e1, and cyp3) and downregulated the phase II enzyme genes (gst1, gpx, gsr2, gclc, and nqo1), resulting in increased ROS levels and cytotoxic effects. MC-LR exhibited cytotoxicity, reducing cell viability and inducing abnormal nuclear morphology with a 48 h-IC50 value of approximately 120 µm. MC-LR exposure caused biochemical changes indicative of oxidative stress damage and evident hepatopancreatic lesions. Additionally, MC-LR exposure regulated the levels of bax and bcl-2 expression, activating caspase 3 and 6 to induce cell apoptosis. Intervention with NAC attenuated MC-LR-induced ROS production and associated toxic effects. Transcriptome analysis revealed enrichment of differentially expressed genes in pathways related to cytochrome P450-mediated xenobiotic metabolism and the FoxO signaling pathway. These findings shed light on the potential mechanisms underlying MC-LR toxicity and provide valuable references for further research and conservation efforts regarding the health of aquatic animals.

Resveratrol ameliorates intestinal lipid metabolism through the PPAR signaling pathway in high-fat diet-fed red tilapia (Oreochromis niloticus).

Feeding high-fat (HF) diets has been shown to cause hepatic and intestinal impairment in fish species, but the mode of action, especially the pathways involved in the intestine, has not been determined yet. In this study, the effects of resveratrol (RES) supplementation on the intestinal structure, microbial flora, and fat metabolism in red tilapia (Oreochromis niloticus) were determined. The results showed RES maintained the structural integrity of the intestine and significantly increased the number of goblet cells in the midgut. RES significantly induced interferon (IL)-1ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α, serumal and fecal trimetlylamine oxide (TMAO) and lipopolysaccharides (LPS), intestinal acetic acid levels. However, the concentrations of bound bile acids increased in HF-fed red tilapia. Atp5fa1 and Pafah1b3 significantly increased, Pmt and Acss2 significantly decreased, respectively, with RES supplementation, which was alleviated and retained at the same level in the selisistat (EX527) group. While for transcriptome and proteomics results, RES was found to promote fatty acid ß-oxidation and arachidonic acid metabolism associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway. The next validation experiment showed some genes related to apoptosis and fatty acid metabolism pathways were altered by RES supplementation. Namely, sn6, loc100702698, new_14481, and prkaa1 were upregulated, while ffrs1, ap3s1, and loc100705861 were downregulated. RES significantly increased Planctomycetes and Verrucomicrobia while decreased Moonvirus, Citrobacter, and Pseudomonas. Akkermansia and Fusobacterium significantly increased and Aeromonas significantly decreased. Thus, unsaturated fatty acid biosynthesis significantly increased and carbohydrate/energy metabolism decreased. To conclude, RES enabled the body to complete fatty acid ß-oxidation and arachidonic acid metabolism, whereas the addition of inhibitors increased the expression of the phagosome transcriptome and reduced fatty acid ß-oxidative metabolism.

Effects of prometryn on oxidative stress, immune response and apoptosis in the hepatopancreas of Eriocheir sinensis (Crustacea: Decapoda).

Prometryn, a triazine pesticide product used to control weed growth, poses a high risk to aquatic organisms in the environment. Several toxicological evaluations have been performed on bony fish and shrimp exposed to prometryn. However, there have been no reports conducted on the toxic mechanism of prometryn with regard to Eriocheir sinensis. In this study, our research evaluated the toxic effects of prometryn via in vitro and in vivo toxicity tests on E. sinensis. Firstly, we estimated the exposure toxicity of prometryn to E. sinensis, and then we constructed a 6 h transcriptional profile and conducted an enrichment analysis. To further reveal the toxicity of prometryn, the hepatopancreas (hepatopancreatic cells) was analyzed for antioxidant, immune and lipid-metabolism-related enzymes, antioxidant- and apoptosis-related gene expression, histopathology and TUNEL. From the results, we determined that the 96 h-LD50 was 70.059 mg/kg, and using RNA-seq, we identified 933 differentially expressed genes (DEGs), which were mainly enriched in the amino and fatty acid metabolism and the cell-fate-determination-related signaling pathway. The results of the biochemical assays showed that prometryn could significantly decrease the activities/levels of CAT, SOD, GSH, AKP and ACP, reduce the levels of T-AOC, TG, TCH, C3 and C4, and increase the MDA content. In addition, the expression levels of Nrf2, GSTs and HO-1 were first upregulated and then downregulated with increasing time. Histopathology showed that prometryn damaged the structure of the hepatopancreas cells and induced apoptosis, suggesting that the PI3K-Akt signaling pathway may be involved in the damage process of hepatopancreas cells (PI3K, PDK and Akt were downregulated whereas Bax was upregulated), leading to their apoptosis. The above results indicated that prometryn could cause injury of the hepatopancreas through oxidative stress, induce cell apoptosis, disrupt the lipid metabolism and cause immune damage. This study provided useful data for understanding and evaluating the toxicity of prometryn to aquatic crustacea.

Salvianolic Acid B Regulates Oxidative Stress, Autophagy and Apoptosis against Cyclophosphamide-Induced Hepatic Injury in Nile Tilapia (Oreochromis niloticus).

Salvianolic acid B (Sal B), as one of the main water-soluble components of Salvia miltiorrhizae, has significant pharmacological activities, including antioxidant, free radical elimination and biofilm protection actions. However, the protective effect of Sal B on Nile tilapia and the underlying mechanism are rarely reported. Therefore, the aim of this study was to evaluate the effects of Sal B on antioxidant stress, apoptosis and autophagy in Nile tilapia liver. In this experiment, Nile tilapia were fed diets containing sal B (0.25, 0.50 and 0.75 g·kg-1) for 60 days, and then the oxidative hepatic injury of the tilapia was induced via intrapleural injection of 50 g·kg-1 cyclophosphamide (CTX) three times. After the final exposure to CTX, the Nile tilapia were weighed and blood and liver samples were collected for the detection of growth and biochemical indicators, pathological observations and TUNEL detection, as well as the determination of mRNA expression levels. The results showed that after the CTX treatment, the liver was severely damaged, the antioxidant capacity of the Nile tilapia was significantly decreased and the hepatocyte autophagy and apoptosis levels were significantly increased. Meanwhile, dietary Sal B can not only significantly improve the growth performance of tilapia and effectively reduce CTX-induced liver morphological lesions, but can also alleviate CTX-induced hepatocyte autophagy and apoptosis. In addition, Sal B also significantly regulated the expression of genes related to antioxidative stress, autophagy and apoptosis pathways. This suggested that the hepatoprotective effect of Sal B may be achieved through various pathways, including scavenging free radicals and inhibiting hepatocyte apoptosis and autophagy.

Alleviative effects of astragaloside IV on cyclophosphamide-induced oxidative damage and immunosuppression in tilapia (Oreochromis niloticus).

Astragaloside IV (ASIV) has effects of antioxidation and immunologic enhancement. However, there are few reports on the application and potential mechanism of ASIV in aquaculture. In this study, we investigated the effect of ASIV on growth, antioxidation, and immune function of tilapia. Tilapia were fed a diet containing 0.1, 0.2, and 0.5 g·kg-1 ASIV for 60 days, followed by an intrapleural injection of 50 mg·kg-1 cyclophosphamide (CTX) to induce oxidative damage and immunosuppression. Then tilapia were weighed and blood, liver, spleen, kidney, and intestinal were collected. The results showed ASIV increased the final weight, relative weight rate, and specific growth rate of tilapia, reduce conversion ratio, and reduced the morphological lesions of tissues. Meanwhile, ASIV alleviated CTX-induced oxidative damage by improving antioxidant activity in serum and tissues and inhibiting lipid peroxidation. Additionally, ASIV attenuated the immunosuppression of tilapia caused by CTX, regulated immunochemical indexes in serum, increased the viability of peripheral blood leukocytes and head kidney macrophages, and restored respiratory burst activity (O2-) in head kidney macrophages and splenocytes. Furthermore, qPCR data showed ASIV up-regulated antioxidant-related gene expression of nrf2, ho-1, gpx3, and cat and immune-related gene expression including C3 and igm. In conclusion, ASIV as a feed additive can not only improve the growth performance but also enhance the antioxidant capacity and immune function of tilapia, which may be associated with the ability of ASIV to scavenge free radicals, reduce lipid peroxidation levels, and stabilize numbers of immune cells.

Microcystin-LR induces apoptosis in Juvenile Eriocheir sinensis via the mitochondrial pathway.

Microcystin-LR (MC-LR), the toxic substance of cyanobacteria secondary metabolism, widely exists in water environments and poses great risks to living organisms. Some toxicological assessments of MC-LR have performed at physiological and biochemical levels. However, plenty of blanks about the potential mechanism in aquatic crustacean remains. In this study, we firstly assessed the exposure toxicity of MC-LR to juvenile E. sinensis and clarified that the 96 h LD50 of MC-LR was 73.23 µg/kg. Then, hepatopancreas transcriptome profiles of MC-LR stressed crabs were constructed at 6 h post-injection and 37 differential expressed genes (DEGs) were identified. These DEGs were enriched in cytoskeleton, peroxisome and apoptosis pathways. To further reveal the toxicity of MC-LR, oxidative stress parameters (SOD, CAT, GSH-px and MDA), apoptosis genes (caspase 3, bcl-2 and bax) and apoptotic cells were detected. Significant accumulated MDA and rise-fall enzyme activities verified the oxidative stress caused by MC-LR. It is noteworthy that quantitative real-time PCR and TUNEL assay indicated that MC-LR stress-induced apoptosis via the mitochondrial pathway. Interestingly, activator protein-1 may play a crucial role in mediating the hepatotoxicity of MC-LR by regulating apoptosis and oxidative stress. Taken together, our study investigated the toxic effects and the potential molecular mechanisms of MC-LR on juvenile E. sinensis. It provided useful data for exploring the toxicity of MC-LR to aquatic crustaceans at molecular levels.

Optimum feeding frequency of juvenile largemouth bass (Micropterus salmoides) reared in in-pond raceway recirculating culture system.

This study was conducted to determine the effects of feeding frequency on the growth, serum biochemical parameters, antioxidant status and hepatic growth hormone (GH), insulin-like growth factor I (IGF-I), lipoprotein lipase (LPL) and hepatic lipase (HL) gene expression levels of juvenile largemouth bass (Micropterus salmoides) reared in an in-pond raceway recirculating culture system (IPRS). Fish (initial body weight 5.0 ± 0.4 g) were hand-fed with a commercial diet under one of three different feeding frequency treatments (2, 3 or 4 meals/day) for 120 days. The results indicated that no significant differences were observed in the final body weight, weight gain and specific growth rate of fish fed different feeding frequencies on 30 days and 60 days (P > 0.05). Fish fed 2 times/day had higher growth than that fed 4 times/day on 90 days but had higher growth than those fed 3 and 4 times/day on 120 days. No significant differences were found in serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities, total protein (TP), lysozyme and triglyceride (TG) content, hepatic total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) activities and malondialdehyde (MDA) content among fish fed different feeding frequency (P > 0.05). Serum glucose (Glu) content and catalase (CAT) activity decreased, while total cholesterol (TC) content increased with increasing feeding frequency. Fish fed 2 times/day had higher hepatic total superoxide dismutase (T-SOD) than that fed 4 times/day on 60 days, 90 days and 120 days (P < 0.05). Fish fed 2 times/day had higher IGF-1 gene mRNA expression on 30 days, 60 days and 120 days (P < 0.05), while no significant difference on 90 days. No significant difference was found in GH gene mRNA expression on 30 days and 60 days, while fish fed 4 times/day had lower values than that fed 2 times/day on 90 days and 120 days (P < 0.05). Fish fed 2 times/day had significantly higher LPL mRNA expression level than that fed 4 times/day on 60 days and 90 days and had significantly higher HL mRNA expression level on 60 days, 90 days and 120 days (P < 0.05). Based on growth, physiology, hepatic gene expression levels, labour costs and intensity, the optimal feeding frequency of largemouth bass (average body weight 5.0 ± 0.4 g) reared in IPRS is 2 times/day. These data are very necessary for the optimizing of culture conditions and feeding management strategy in IPRS culture operations.

Genome and population sequencing of a chromosome-level genome assembly of the Chinese tapertail anchovy (Coilia nasus) provides novel insights into migratory adaptation.

BACKGROUND: Seasonal migration is one of the most spectacular events in nature; however, the molecular mechanisms related to this phenomenon have not been investigated in detail. The Chinese tapertail, or Japanese grenadier anchovy, Coilia nasus, is a valuable migratory fish of high economic importance and special migratory dimorphism (with certain individuals as non-migratory residents). RESULTS: In this study, an 870.0-Mb high-quality genome was assembled by the combination of Illumina and Pacific Biosciences sequencing. Approximately 812.1 Mb of scaffolds were linked to 24 chromosomes using a high-density genetic map from a family of 104 full siblings and their parents. In addition, population sequencing of 96 representative individuals from diverse areas along the putative migration path identified 150 candidate genes, which are mainly enriched in 3 Ca2+-related pathways. Based on integrative genomic and transcriptomic analyses, we determined that the 3 Ca2+-related pathways are critical for promotion of migratory adaption. A large number of molecular markers were also identified, which distinguished migratory individuals and non-migratory freshwater residents. CONCLUSIONS: We assembled a chromosome-level genome for the Chinese tapertail anchovy. The genome provided a valuable genetic resource for understanding of migratory adaption and population genetics and will benefit the aquaculture and management of this economically important fish.

Monogonont Rotifer, Brachionus calyciflorus, Possesses Exceptionally Large, Fragmented Mitogenome.

In contrast to the highly conserved mitogenomic structure and organisation in most animals (including rotifers), the two previously sequenced monogonont rotifer mitogenomes were fragmented into two chromosomes similar in size, each of which possessed one major non-coding region (mNCR) of about 4-5 Kbp. To further explore this phenomenon, we have sequenced and analysed the mitogenome of one of the most studied monogonont rotifers, Brachionus calyciflorus. It is also composed of two circular chromosomes, but the chromosome-I is extremely large (27 535 bp; 3 mNCRs), whereas the chromosome-II is relatively small (9 833 bp; 1 mNCR). With the total size of 37 368 bp, it is one of the largest metazoan mitogenomes ever reported. In comparison to other monogononts, gene distribution between the two chromosomes and gene order are different and the number of mNCRs is doubled. Atp8 was not found (common in rotifers), and Cytb was present in two copies (the first report in rotifers). A high number (99) of SNPs indicates fast evolution of the Cytb-1 copy. The four mNCRs (5.3-5.5 Kb) were relatively similar. Publication of this sequence shall contribute to the understanding of the evolutionary history of the unique mitogenomic organisation in this group of rotifers.