Effects of effective microorganisms on the physiological status, intestinal microbiome, and serum metabolites of Eriocheir sinensis.

The compound known as effective microorganisms (EMs) is widely used in aquaculture to improve water quality, but how they affect the health of Chinese mitten crab (Eriocheir sinensis) is unclear, especially in terms of intestinal microbiota and serum metabolites. In this study, we fed juvenile crabs with an EM-containing diet to explore the effects of EM on the physiological status, intestinal microbiome, and metabolites of E. sinensis. The activities of alanine aminotransferase and alkaline phosphatase were significantly enhanced by EM, indicating that EM supplementation effectively enhanced the antioxidant capacity of E. sinensis. Proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, and Actinobacteria were the main intestinal microbes in both the control and EM groups. Linear discriminant effect size analysis showed that Fusobacteriaceae, Desulfovibrio, and Morganella were biomarkers in the control group, and Exiguobacterium and Rhodobacteraceae were biomarkers in the EM group. Metabolomics analysis revealed that EM supplementation increased cellular energy sources and decreased protein consumption, and oxidative stress. Together, these results indicate that EM can optimize the intestinal microbiome and serum metabolites, thereby benefiting the health of E. sinensis.

Microcystin-LR-induced autophagy via miR-282-5p/PIK3R1 pathway in Eriocheir sinensis hepatopancreas.

With the intensifying climate warming, blue-green algae blooms have become more frequent and severe, releasing environmental hazards such as microcystin that pose potential threats to human and animal health. Autophagy has been shown to play a crucial role in regulating immune responses induced by environmental hazards, enabling cells to adapt to stress and protect against damage. Although microcystin-LR (MC-LR) has been identified to affect autophagy in mammalian, its impact on aquatic animals has been poorly studied. To investigate the toxicological effects of MC-LR in aquatic ecosystems, we constructed a microRNA profile of acute MC-LR stress in the hepatopancreas of the Chinese mitten crab. Interestingly, we found the MC-LR exposure activated autophagy in the hepatopancreas based on the following evidence. Specifically, mRNA expression level of ATG7, Beclin1 and Gabarap was significantly up-regulated, autophagy regulatory pathways were significantly enriched, and numerous autolysosomes and autophagosomes were observed. Additionally, we found that miR-282-5p and its target gene PIK3R1 played important regulatory roles in autophagy by in vivo and in vitro experiments. Overexpression of miR-282-5p mimicked MC-LR-induced autophagy by inhibiting PIK3R1 expression, while miR-282-5p silencing inhibited autophagy by promoting PIK3R1 expression. Altogether, our findings suggest that MC-LR increases miR-282-5p, which then targets inhibition of PIK3R1 to stimulate autophagy. This study focused on the stress response regulatory mechanisms of juvenile crabs to toxic pollutants in water, offering a potential target for alleviating the toxicity of MC-LR. These findings lay a foundation for reducing the toxicity of MC-LR and environmental hazards in organisms.

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.

Integrated Analysis of Transcriptomics and Metabolomics Unveil the Novel Insight of One-Year-Old Precocious Mechanism in the Chinese Mitten Crab, Eriocheir sinensis.

Eriocheir sinensis is traditionally a native high-value crab that is widely distributed in eastern Asia, and the precocity is considered the bottleneck problem affecting the development of the industry. The precocious E. sinensis is defined as a crab that reaches complete sexual maturation during the first year of its lifespan rather than as normally in the second year. However, the exact regulatory mechanisms underlying the precocity are still unclear to date. This study is the first to explore the mechanism of precocity with transcriptome-metabolome association analysis between the precocious and normal sexually mature E. sinensis. Our results indicated that the phenylalanine metabolism (map00360) and neuroactive ligand-receptor interaction (map04080) pathways play an important role in the precocity in the ovary of E. sinensis. In map00360, the predicted aromatic-L-amino-acid decarboxylase and 4-hydroxyphenylpyruvate dioxygenase isoform X1 genes and the phenethylamine, phenylethyl alcohol, trans-2-hydroxycinnamate, and L-tyrosine metabolites were all down-regulated in the ovary of the precocious E. sinensis. The map04080 was the common KEGG pathway in the ovary and hepatopancreas between the precocious and normal crab. In the ovary, the predicted growth hormone secretagogue receptor type 1 gene was up-regulated, and the L-glutamate metabolite was down-regulated in the precocious E. sinensis. In the hepatopancreas, the predicted forkhead box protein I2 gene and taurine metabolite were up-regulated and the the L-glutamate metabolite was down-regulated in the precocious crab. There was no common pathway in the testis. Numerous common pathways in the hepatopancreas between male precocious and normal crab were identified. The specific amino acids, fatty acids and flavorful nucleotide (inosine monophosphate (MP), cytidine MP, adenosine MP, uridine MP, and guanosine MP) contents in the hepatopancreas and gonads further confirmed the above omics results. Our results suggest that the phenylalanine metabolism may affect the ovarian development by changing the contents of the neurotransmitter and tyrosine. The neuroactive ligand-receptor interaction pathway may affect the growth by changing the expressions of related genes and affect the umami taste of the gonads and hepatopancreas through the differences of L-glutamate metabolite in the precocious E. sinensis. The results provided valuable and novel insights on the precocious mechanism and may have a significant impact on the development of the E. sinensis aquaculture industry.

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.

Effects of Submerged Macrophytes on the Growth, Morphology, Nutritional Value, and Flavor of Cultured Largemouth Bass (Micropterus salmoides).

Aquaculture environment plays important roles in regulating the growth, morphology, nutrition, and flavor of aquatic products. The present study investigated growth, morphology, nutrition, and flavor formation in largemouth bass (Micropterus salmoides) cultured in the ponds with (EM group) and without (M group) the submerged macrophytes (Elodea nuttallii). Fish in the EM group showed a significantly greater body length, higher growth rate, and lower hepatosomatic index than those in the M group (p< 0.05). Moreover, compared with fish in the M group, those in the EM group showed improved muscle quality with significantly elevated levels of crude protein, total free and hydrolysable amino acids, and polyunsaturated fatty acids (p < 0.05). Specifically, certain amino acids related to flavor (Glu, Asp, Ala, and Arg) and valuable fatty acids (C18:2, C18:3n3, C20:3n3, and C22:6) were more abundant in the EM group (p < 0.05). In addition, the levels of 19 volatile (p < 0.05) were significantly higher in the EM group than in the M group. Therefore, E. nuttallii significantly improved growth, morphological traits, nutritional components, and characteristic flavor in largemouth bass, indicating the superior nutritional value and palatability of fish cultured with submerged macrophytes.

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.

Geographical and temporal variation of multiple paternity in invasive mosquitofish (Gambusia holbrooki, Gambusia affinis).

Multiple paternity (MP) increases offspring's genetic variability, which could be linked to invasive species' evolvability in novel distribution ranges. Shifts in MP can be adaptive, with greater MP in harsher/colder environments or towards the end of the reproductive season, but climate could also affect MP indirectly via its effect on reproductive life histories. We tested these hypotheses by genotyping N = 2,903 offspring from N = 306 broods of two closely related livebearing fishes, Gambusia holbrooki and Gambusia affinis. We sampled pregnant females across latitudinal gradients in their invasive ranges in Europe and China, and found more sires per brood and a greater reproductive skew towards northern sampling sites. Moreover, examining monthly sampling from two G. affinis populations, we found MP rates to vary across the reproductive season in a northern Chinese, but not in a southern Chinese population. While our results confirm an increase of MP in harsher/more unpredictable environments, path analysis indicated that, in both cases, the effects of climate are likely to be indirect, mediated by altered life histories. In both species, which rank amongst the 100 most invasive species worldwide, higher MP at the northern edge of their distribution probably increases their invasive potential and favours range expansions, especially in light of the predicted temperature increases due to global climate changes.

Responses of gonadal transcriptome and physiological analysis following exposure to 17α-ethynylestradiol in adult rare minnow Gobiocypris rarus.

17α-ethynylestradiol (EE2), a synthetic estrogen commonly used in the oral contraceptive pills, disrupts the sexual differentiation, gonadal development and reproduction in aquatic species. Nowadays aquatic species and even humans still have the potential risks of exposure to EE2. However, the mechanism of EE2 endocrine disruption is still unclear. Aiming to elucidate molecular mechanisms, we analyzed transcriptome profiling of gonads, gonadal histology and the sex steroid hormones in response to EE2 in G. rarus. Through this study, we obtained eight RNA-Seq libraries upon EE2 exposure, and found some key genes and pathways in correlation with the disruption effects of EE2. We found EE2 could disrupt oocyte development and spermatogenesis in adult G. rarus, and EE2 has more obvious disruption effects on male G. rarus than females. Interestingly, EE2 was indicated to be an exogenous DPC-inducing agent and ppp2r3b was suggested to be a spermatogenesis candidate gene in rare minnow. The differential gene expressions of rps30, samp9, ppp2r3b and spartan upon EE2 exposure suggest EE2's disruption effects on gonads could attribute to altered pathways of translation, ribosome biogenesis and cell division.

Molecular identification of Kiss/GPR54 and function analysis with mRNA expression profiles exposure to 17α-ethinylestradiol in rare minnow Gobiocypris rarus.

17α-ethinylestradiol (EE2) is a widely existed endocrine disrupting chemical in water environment. Kisspeptins act as indispensable regulators through GPR54 in the hypothalamic-pituitary-gonadal (HPG) axis. This study aimed to provide further understanding of the effect of EE2 on HPG axis. Molecular cloning and tissue distribution of kiss genes and GPR54s were performed in Gobiocypris rarus. The mRNA expression profiles of kiss1, kiss2, GPR54s and GnRHs were detected in G. rarus brain and/or gonad following 3- and 6-days EE2 (1, 5, 25 and 125 ng/L) exposure. Results showed that kiss genes and GPR54s were highly expressed in brain and gonad. Both kiss1 and kiss2 were increased in female brain and suppressed in male brain following EE2 exposure. GnRHs were inhibited in a concentration-dependent manner in male brain following 3-days EE2 exposure. In gonad, GPR54b was almost suppressed in all of EE2 concentrations. The present findings suggest that EE2 impacts the genes expression of Kiss/GPR54-GnRH system in G. rarus, thereby probably disturbing the neuroendocrine homeostasis.

Oxidative stress and immunotoxic effects of bisphenol A on the larvae of rare minnow Gobiocypris rarus.

Bisphenol A (BPA), a known endocrine disrupting chemical, is ubiquitous in the aquatic environment and can pose risk to the health of aquatic organisms. Studies on immunotoxicity of BPA in aquatic organisms are limited. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to 1, 225 and 1000µg/L BPA for 7 days. Inflammatory effects of BPA exposure were assessed from the increased production of nitric oxide (NO) and reactive oxygen species (ROS), the change of iNOS mRNA and other TLRs-associated immune gene expression. Our findings provide evidences that different concentrations of BPA can induce a toxic response in fish to produce reactive free radicals which can affect the function of T lymphocytes and decrease the transcription levels of cytokine genes. The excess production of H2O2, induced oxidative stress and suppressed TLR4/NF-κB signaling, leading to immunosuppressive effects in fish larvae. The present results suggest that BPA has the potential to induce oxidative stress accompanied by immunosuppression in rare minnow larvae.

Molecular mechanism of endocrine system impairment by 17α-methyltestosterone in gynogenic Pengze crucian carp offspring.

The effects of synthetic androgen 17α-methyltestosterone (MT) on endocrine impairment were examined in crucian carp. Immature 7-month old mono-female Pengze crucian carp (Pcc) F2 offspring were exposed to 50 and 100 µg/L of MT (week 2, 4, and 8). Gonadosomatic index, hepatosomatic index and intestine weight altered considerably and oocyte development was repressed. In the treatment groups, ovarian 11-ketotestosterone decreased, whereas 17ß-estradiol and testosterone increased, and ovarian aromatase activities increased at week 4. However, in the brain tissue, those values significantly decreased. Quantitative RT-PCR analysis demonstrated changes in steroid receptor genes and upregulation of steroidogenic genes (Pcc-3bhsd, Pcc-11bhsd2 Pcc-cyp11a1), while the other three steroidogenic genes (Pcc-cyp17a1, Pcc-cyp19a1a and Pcc-star) decreased from week 4 to week 8. Ovarian, hepatic Pcc-vtg B and vitellogenin concentration increased in both 50 and 100 µg/L of MT exposure groups. This study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, which suggests that MT could directly target estrogen signaling pathway, or indirectly affect steroidogenesis and vitellogenesis.

Molecular characterization of PXR and two sulfotransferases and hepatic transcripts of PXR, two sulfotransferases and CYP3A responsive to bisphenol A in rare minnow Gobiocypris rarus.

Bisphenol A (BPA), a wide distributed endocrine-disrupting chemical, has attracted many attentions. To explore the effect of BPA on hepatic metabolic pathways in Gobiocypris rarus, full-length cDNAs of pregnane X receptor (PXR) and two sulfotransferases (SULT1 ST4 and SULT1 ST6) were firstly isolated and characterized. We detected tissues distribution of PXR, CYP3A, SULT1 ST4 and SULT1 ST6 in adult G. rarus. Then we investigated hepatic transcript profiles of these four genes in adult G. rarus exposed to BPA at concentrations of 5, 15, and 50 µg/L for 14 and 35 days. It demonstrates that these four genes are all highly expressed in liver of both male and female adult G. rarus. In response to BPA, sexual dimorphism of expression patterns for PXR, CYP3A, and SULT1 ST6 shows in G. rarus, which includes increase of mRNA levels in females and decrease of mRNA levels in males in both exposure durations of 14 and 35 days. SULT1 ST6 mRNA demonstrates high responsiveness to BPA in both genders and we recommended SULT1 ST6 as a candidate biomarker for BPA exposure.

Responsiveness of four gender-specific genes, figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol in adult rare minnow Gobiocypris rarus.

Proteins encoded by figla, foxl2, scp3 and sox9a play important roles in gonad differentiation and reproduction. In the present study, we aimed to determine the responsiveness of figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol (EE2) in the gonads of adult Gobiocypris rarus. Full-length cDNAs of figla, scp3 and sox9a were cloned and characterized by RT-PCR and RACE methods. Expression patterns in adult tissues were investigated. Results indicated that figla was predominantly expressed in adult ovaries and scp3 was restrictively expressed in the male testes and sox9a was principally expressed in the brains of both genders and the testes of males. Gene expression profiles of figla, foxl2, scp3 and sox9a were analyzed in the gonads of adult G. rarus exposed to EE2 at 1, 5, 25, and 125ng/L for 3 and 6days. Three-day EE2 treatment at 1-125ng/L all caused a significant increase of figla transcript in testes and foxl2 transcript in ovaries. However, six-day EE2 exposure at 1-125ng/L repressed figla and scp3 transcript in testes and foxl2 transcript in ovaries. The present study indicates that the testicular transcripts of figla and scp3 in males and the ovarian foxl2 transcript in females have high responsiveness to EE2 and they can be used as sensitive molecular biomarkers for early warning to monitor the environmental estrogenic chemicals in fresh water environment. The present study also suggests that the effective EE2 dosage for feminization in male G. rarus might be at least 25ng/L.

Induction of oxidative stress and the transcription of genes related to apoptosis in rare minnow (Gobiocypris rarus) larvae with Aroclor 1254 exposure.

Polychlorinated biphenyls (PCBs) are a group of environmental contaminants widely dispersed in aquatic system. Recent data have shown that Aroclor 1254 (a highly chlorinated PCB mixture) has the potential to induce oxidative stress. The antioxidant genes are usually up-regulated in response to the oxidative stress. However, the mechanisms underlying the modulation are little known. We hypothesized that nuclear factors erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB) might be involved in the regulation. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to the Aroclor 1254 at four concentrations (5, 50, 500 and 5000µg/L) for 7 days. We found that the mRNA expressions of antioxidant genes (Cu/Zn-sod, Mn-sod, Cat, Gpx1 and Gclc) were strongly enhanced by Aroclor 1254 at high concentrations. H2O2 was significantly induced by 500 and 5000µg/L Aroclor 1254 exposure and protein thiol significantly decreased with 5000µg/L Aroclor 1254 exposure. The expression of Nrf2 and NF-κB were significantly up-regulated. Taken together, we proposed that the activation of Nrf2 and NF-κB by ROS might be a potential mechanism underlying the antioxidant gene expression induction in G. rarus larvae by Aroclor 1254. Furthermore, we investigated that the expression of genes related with apoptosis. The gene expression patterns reveal that waterborne Aroclor 1254-induced apoptosis is probably through DNA damage (p53 and p53 upregulated modulator of apoptosis (Puma)), disrupting mitochondrial membrane integrity (B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax)) and c-jin N-terminal kinase (JNK)-mediated apoptotic pathway (thioredoxin 1 (Trx1) and c-jun N-terminal kinase (JNK)). Aroclor 1254 at 5µg/L did not cause any changes in G. rarus. The findings will help us to understand the toxicological mechanism of Aroclor 1254 in fish and properly assess the risk of the environmental contaminant.

Prometryn exposure disrupts the intestinal health of Eriocheir sinensis: Physiological responses and underlying mechanism.

Most toxicity studies of prometryn in non-target aquatic animals have focused on hepatotoxicity, cardiotoxicity, embryonic developmental and growth toxicity, while studies on the molecular mechanisms of intestinal toxicity of prometryn are still unknown. In the current study, the intestinal tissues of the Chinese mitten crab (Eriocheir sinensis) were used to uncover the underlying molecular mechanisms of stress by 96-h acute in vivo exposure to prometryn. The results showed that prometryn activated the Nrf2-Keap1 pathway and up-regulated the expression of downstream antioxidant genes. Prometryn induced the expression of genes associated with non-specific immunity and autophagy, and induced apoptosis through the MAPK pathway. Interestingly, the significant up-or down-regulation of the above genes mainly occurred at 12 h- 24 h after exposure. Intestinal flora sequencing revealed that prometryn disrupted the intestinal normal barrier function mainly by reducing beneficial bacteria abundance, which further weakened the intestinal resistance to exogenous toxicants and caused an inflammatory response. Correlation analyses found that differential flora at the genus level had potential associations with gut stress-related genes. In conclusion, our study contributes to understanding the molecular mechanisms behind the intestinal stress caused by herbicides on aquatic crustaceans.

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.

Characterization of four nr5a genes and gene expression profiling for testicular steroidogenesis-related genes and their regulatory factors in response to bisphenol A in rare minnow Gobiocypris rarus.

Bisphenol A (BPA) widely used in the manufacture of numerous products is ubiquitous in aquatic environment. To explore the mechanisms of BPA-mediated actions, male rare minnow Gobiocypris rarus were exposed to BPA at concentrations of 5, 15, and 50 µg/L for 14 and 35 days in the present study. Four subtypes of nr5a gene encoding important transcription factors for steroidogenesis were characterized, and tissue distribution analysis demonstrated distinct expression profiling of the four genes in G. rarus. BPA at environmentally relevant concentration (5 µg/L) caused increase of gonadosomatic index (GSI) of male fish. In response to BPA, no obvious changes on the testis development were observed. Modulation of vtg mRNA expression by BPA suggests estrogenic and/or anti-estrogenic effects of BPA were dependent on exposed duration (14 or 35 days). Gene expression profiling for testicular steroidogenesis-related genes, sexual steroid receptors, gonadotropin receptors, and transcription factors indicates differential regulation was dependent on exposure duration and dose of BPA. The correlation analysis at mRNA level demonstrates that the BPA-mediated actions on testicular steroidogenesis might involve sex steroid hormone receptor signaling, gonadotropin/gonadotropin receptor pathway, and transcription factors such as nuclear receptor subfamily 5, group A (Nr5a), fork head box protein L2 (Foxl2).

Oxidative stress and immune response of hepatopancreas in Chinese mitten crab Eriocheir sinensis under lipopolysaccharide challenge.

Chinese mitten crab (Eriocheir sinensis; H. Milne Edwards, 1853) is one of the important farmed crustaceans in China. Lipopolysaccharide (LPS), as a harmful factor, is prone to occur during the farming process of crabs. Aiming to test the hypothesis that damage degrees of the hepatopancreas in E. sinensis is correlated to LPS concentrations, in this study, E. sinensis were injected with LPS (50 µg/kg, and 500 µg/kg) and analyzed for the activity of antioxidant and immune-related enzymes, immune-related gene expression, and histopathological of hepatopancreas. As result, the hepatopancreas of E. sinensis immune-related genes, i.e., Dorsal, HSP90, Toll2, TLRs, Tube, and proPO, were significantly affected by LPS challenge. Among immune-related genes, Dorsal and proPO might play key roles in combating the LPS challenge. The activity of CAT gradually decreased with the increase of time, and the total antioxidant capacity was decreased after LPS challenge, indicating the inhibition of LPS on the antioxidant system. Interestingly, the decreasing trend of AKP and ACP activity suggested the immune system of crabs was affected by LPS challenge. The hepatopancreas section showed that the damage degree of hepatopancreas was different under the challenge of LPS with different concentrations, and the damage degree was proportional to the concentration. Our findings provide useful information for understanding the mechanism of hepatopancreas injury of E. sinensis induced by LPS infection.

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.