Chronic cypermethrin induced toxicity and molecular fate assessment within common carp (Cyprinus carpio) using multiple biomarkers approach and its novel therapeutic detoxification.

Cypermethrin (CYP) is a chemical of emerging concern which has persistent and bioaccumulating impacts as it can be found extensively in freshwater ecosystem and agricultural products. It has exposure risk and toxic effects over human edible fish, as common carp. Four groups were designed for toxicity assessment and detoxification approach: control group (CL), CYP exposure group (CYP), CYP + 10% M. oleifera leaves and 10% M. oleifera seeds (CMO group), 10% M. oleifera leaves and 10% M. oleifera seeds (MO group). Trial period was forty days during which cohort of 240 fish in CYP and CMO group was exposed to 1/5 of 96h LC50 of CYP (0.1612 µg/L). CYP-exposed carp exhibited lower growth parameters, but carp fed with 10% M. oleifera seeds and leaves showed significant improvement in growth rate (SGR, RGR) and weight gain (WG) as compared to the control group. CYP exposure negatively affected haemato-biochemical parameters. Moreover, CYP exposure also led to oxidative stress, damaged immunological parameters, genotoxicity and histopathological damage in liver and intestinal cells. Whereas, M. oleifera supplementation has ameliorated these conditions. Thereby, supplementation with M. oleifera is potential and novel therapeutic detoxication approach for common carp and human health against persistent and bioaccumulating emerging chemicals.

Dietary sodium acetate and sodium butyrate improve high-carbohydrate diet utilization by regulating gut microbiota, liver lipid metabolism, oxidative stress, and inflammation in largemouth bass (Micropterus salmoides).

BACKGROUND: Adequate level of carbohydrates in aquafeeds help to conserve protein and reduce cost. However, studies have indicated that high-carbohydrate (HC) diet disrupt the homeostasis of the gut-liver axis in largemouth bass, resulting in decreased intestinal acetate and butyrate level. METHOD: Herein, we had concepted a set of feeding experiment to assess the effects of dietary sodium acetate (SA) and sodium butyrate (SB) on liver health and the intestinal microbiota in largemouth bass fed an HC diet. The experimental design comprised 5 isonitrogenous and isolipidic diets, including LC (9% starch), HC (18% starch), HCSA (18% starch; 2 g/kg SA), HCSB (18% starch; 2 g/kg SB), and HCSASB (18% starch; 1 g/kg SA + 1 g/kg SB). Juvenile largemouth bass with an initial body weight of 7.00 ± 0.20 g were fed on these diets for 56 d. RESULTS: We found that dietary SA and SB reduced hepatic triglyceride accumulation by activating autophagy (ATG101, LC3B and TFEB), promoting lipolysis (CPT1α, HSL and AMPKα), and inhibiting adipogenesis (FAS, ACCA, SCD1 and PPARγ). In addition, SA and SB decreased oxidative stress in the liver (CAT, GPX1α and SOD1) by activating the Keap1-Nrf2 pathway. Meanwhile, SA and SB alleviated HC-induced inflammation by downregulating the expression of pro-inflammatory factors (IL-1ß, COX2 and Hepcidin1) through the NF-κB pathway. Importantly, SA and SB increased the abundance of bacteria that produced acetic acid and butyrate (Clostridium_sensu_stricto_1). Combined with the KEGG analysis, the results showed that SA and SB enriched carbohydrate metabolism and amino acid metabolism pathways, thereby improving the utilization of carbohydrates. Pearson correlation analysis indicated that growth performance was closely related to hepatic lipid deposition, autophagy, antioxidant capacity, inflammation, and intestinal microbial composition. CONCLUSIONS: In conclusion, dietary SA and SB can reduce hepatic lipid deposition; and alleviate oxidative stress and inflammation in largemouth bass fed on HC diet. These beneficial effects may be due to the altered composition of the gut microbiota caused by SA and SB. The improvement effects of SB were stronger than those associated with SA.

Dietary sodium acetate and sodium butyrate attenuate intestinal damage and improve lipid metabolism in juvenile largemouth bass (Micropterus salmoides) fed a high carbohydrate diet by reducing endoplasmic reticulum stress.

High-carbohydrate (HC) diets decrease the intestinal levels of sodium acetate (SA) and sodium butyrate (SB) and impair the gut health of largemouth bass; however, SA and SB have been shown to enhance immunity and improve intestinal health in farmed animals. Thus, the present study was to investigate the effects of dietary SA and SB on HC diet-induced intestinal injury and the potential mechanisms in juvenile largemouth bass. The experiment set five isonitrogenous and isolipidic diets, including a low-carbohydrate diet (9% starch) (LC), a high carbohydrate diet (18% starch) (HC), and the HC diet supplemented with 2 g/kg SA (HCSA), 2 g/kg SB (HCSB) or a combination of 1 g/kg SA and 1 g/kg SB (HCSASB). The feeding experiment was conducted for 8 weeks. A total of 525 juvenile largemouth bass with an initial body weight of 7.00 ± 0.20 g were used. The results showed that dietary SA and SB improved the weight gain rate and specific growth rate (P < 0.05) and ameliorated serum parameters (alkaline phosphatase, acid phosphatase, glutamate transaminase, and glutamic oxaloacetic transaminase) (P < 0.05). And, importantly, dietary SA and SB repaired the intestinal barrier by increasing the expression levels of zonula occludens-1, occludin, and claudin-7 (P < 0.05), reduced HC-induced intestinal damage, and alleviated intestinal inflammation and cell apoptosis by attenuating HC-induced intestinal endoplasmic reticulum stress (P < 0.05). Further results revealed that dietary SA and SB reduced HC-induced intestinal fat deposition by inhibiting adipogenesis and promoting lipolysis (P < 0.05). In summary, this study demonstrated that dietary SA and SB attenuated HC-induced intestinal damage and reduced excessive intestinal fat deposition in largemouth bass.

The protective effect of resveratrol on largemouth bass (Micropterus salmoides) during out-of-season spawning.

In aquaculture production, out-of-season spawning is beneficial to solve the seasonal shortage of fry that are normally produced once annually by species such as largemouth bass (Micropterus salmoides), thereby implementing year-round fry production. Maintaining low temperature over a period of several months can delay largemouth bass ovarian development, but it can cause severe stress to their reproductive function, leading to decreased fertility during out-of-season spawning. Feeding with antioxidants is one of the most effective methods to alleviate the negative effects of low temperature stress. Therefore, the purpose of this study is to: (a) evaluate the changes in oocyte morphology, antioxidant capacity, reproductive hormone-related index, cell apoptosis and autophagy during the out-of-season spawning of largemouth bass, and (b) to investigate the protective effect of the antioxidant resveratrol on this fish during out-of-season spawning from May through August. The study was divided into two groups (three replicates per group, 2000 fish per replicate): control group (Control) (exposure to water temperature of 12-17 °C) and resveratrol supplementation group (Res) (exposure to water temperature of 12-17 °C and fed with 200 mg/kg resveratrol). The results show that: (1) The serum hormones LH and E2 increased first and then remained unchanged, and the ovarian section showed that the ovary remained in stage IV. (2) In the process of off-season reproduction, a large number of follicles experienced follicular atresia, accompanied by endoplasmic reticulum expansion, nuclear chromatin condensation and mitochondrial swelling, which was relieved after feeding resveratrol. (3) Resveratrol decreased the ovarian ROS content and improved the activities of CAT and other antioxidant enzymes in the ovary and liver to some extent. (4) Resveratrol reduced the level of pro-apoptotic (Bax, Caspase3, Caspase8, Caspase9) and autophagy-related components (LC3-B, Beclin-1) while increasing the transcription level of anti-apoptotic (Bcl-2) factors. These findings suggest that resveratrol alleviates some adverse effects of largemouth bass during out-of-season spawning to some extent and provide a model for efficient and high-quality out-of-season spawning.

Exposure to Intermittent Environmental Hypoxia Promotes Vascular Remodeling through Angiogenesis in the Liver of Largemouth Bass (Micropterus salmoides).

In this study, we explored the effects of 4 weeks of intermittent hypoxic exposure (IHE) on liver angiogenesis and related regulatory mechanisms in largemouth bass (Micropterus salmoides). The results indicated that the O2 tension for loss of equilibrium (LOE) decreased from 1.17 to 0.66 mg/L after 4 weeks of IHE. Meanwhile, the red blood cell (RBC) and hemoglobin concentrations significantly increased during IHE. Our investigation also found that the observed increase in angiogenesis was correlated with a high expression of related regulators, such as Jagged, phosphoinositide-3-kinase (PI3K), and mitogen-activated protein kinase (MAPK). After 4 weeks of IHE, the overexpression of factors related to angiogenesis processes mediated by HIF-independent pathways (such as nuclear factor kappa-B (NF-κB), NADPH oxidase 1 (NOX1), and interleukin 8 (IL8)) was correlated with the accumulation of lactic acid (LA) in the liver. The addition of cabozantinib, a specific inhibitor of VEGFR2, blocked the phosphorylation of VEGFR2 and downregulated the expression of downstream angiogenesis regulators in largemouth bass hepatocytes exposed to hypoxia for 4 h. These results suggested that IHE promoted liver vascular remodeling by the regulation of angiogenesis factors, presenting a potential mechanism for the improvement of hypoxia tolerance in largemouth bass.

Chronic hypoxia and Cu2+ exposure induce gill remodeling of largemouth bass through endoplasmic reticulum stress, mitochondrial damage and apoptosis.

Hypoxia and Cu2+ pollution often occur simultaneously in aquatic ecosystems and jointly affect physiology of fish. As the respiratory and ion exchange tissue of fish, how gill responds to the stress induced by these two abiotic environmental factors is still unclear. We have conducted a study by exposing largemouth bass (Micropterus salmoides) to hypoxia (2.0 mg·L-1) and/or Cu2+ (0.5 mg·L-1) for 28 days to answer this question. We subsequently studied respiratory rate, Cu2+ transport, endoplasmic reticulum (ER) stress, mitochondrial damage, and morphology in gill tissue on day 7, 14, 21 and 28. We found that hypoxia exposure increased the respiratory rate of largemouth bass, reflecting the response of largemouth bass to cope with hypoxia. Of note, Cu2+ entered gill by specifically binding to CTR1 and its accumulation dramatically in gill disrupted the response of largemouth bass to hypoxia. Hypoxia and/or Cu2+ exposure led to ER stress and mitochondrial damage in gills of largemouth bass. ER stress and mitochondrial damage induced apoptosis by activating caspase-8 and caspase-9 signaling pathways, respectively. Apoptosis induced by hypoxia and Cu2+ exposure had a positive and synergistic effect on gill remodeling by reducing interlamellar cell masses. In addition, Cu2+ exposure induced hypoxia-like remodeling to gill morphology through mechanisms similar to hypoxia exposure. Most of gene expression changed mainly within 21 days and recovered to the control level on day 28, reflecting the acclimation of largemouth bass to hypoxia and/or Cu2+ exposure at gene expression level. Overall, our research suggests that chronic hypoxia and Cu2+ exposure could induce gill remodeling of largemouth bass through ER stress, mitochondrial damage and apoptosis. The outcomes could provide an insight for fish environmental adaptation and environmental toxicology.

Enhance energy supply of largemouth bass (Micropterus salmoides) in gills during acute hypoxia exposure.

Gills are the location of gas exchange and also the first target organ of fish response for environmental stress. As a multifunctional organ, its energy supply, when faced with insufficient dissolved oxygen in the water, remains unclear. In this study, largemouth bass was subjected to hypoxia stress (1.2 mg/L) for 24 h and 12 h reoxygenation (R12) to evaluate energy supply strategy of gills. Under hypoxia exposure, the respiratory rate of largemouth bass increased by an average of 20 breaths per minute. A total of 2026, 1744, 1003, 579, 485, and 265 differentially expressed genes (DGEs) were identified at 0 h, 4 h, 8 h, 12 h, 24 h, and R12h in gills after hypoxia exposure. KEGG functional analysis of DEGs revealed that the glycolysis/gluconeogenesis pathway was enriched across all the sampling points (0, 4, 8, 12, 24 h, R12). The gene expression and enzyme activity of three rate-limiting enzymes (hexokinase, phosphofructokinase-6, pyruvate kinase) in glycolysis pathway were significantly increased. Increased levels of glycolysis products pyruvate and lactic acid, as well as the number of mitochondria (1.8-fold), suggesting an enhancement of aerobic and anaerobic metabolism of glucose in gills. These results suggest that the gill of largemouth bass enhanced the energy supply during acute exposure to hypoxia stress.

Transcriptome Reveals the Effects of Early Weaning on Lipid Metabolism and Liver Health of Yangtze Sturgeon (Acipenser dabryanus).

The Yangtze sturgeon (Acipenser dabryanus) has recently been declared extinct in the wild, and artificial breeding is the only means to protect its germplasm resources, but it has difficulty in weaning (from live prey to artificial food). In this study, we first performed a histological observation, enzyme-activity determination, and transcriptome sequencing on the livers of juvenile Yangtze sturgeons, and we then cloned five critical genes of lipid metabolism according to the transcriptome-sequencing results. We designed a weaning experiment to analyze their expression levels during weaning. The results showed that the density of hepatocytes and the transaminase activity of the juveniles failed to wean. The differentially expressed genes were enriched significantly in the pathways involving steroid synthesis, amino acid metabolism, and pancreatic secretion. It was found that the mRNA level of the fatty acid-synthesis gene decreased, and the mRNA level of the lipolysis gene increased significantly during weaning. The results of this research indicated that weaning could affect the liver health of Yangtze sturgeon, and it could affect the liver lipid metabolism by inhibiting fatty acid synthesis and promoting lipolysis. This study enhances our understanding of the impact of weaning on the lipid metabolism in fish.

Acute hypoxia promotes the liver angiogenesis of largemouth bass (Micropterus salmoides) by HIF - Dependent pathway.

A 24-h hypoxia exposure experiment was conducted to determine how hypoxia exposure induce liver angiogenesis in largemouth bass. Nitrogen (N2) was pumped into water to exclude dissolved oxygen into 1.2 ± 0.2 mg/L, and liver tissues were sampled during hypoxia exposure of 0 h, 4 h, 8 h, 12 h, 24 h and re-oxygenation for 12 h. Firstly, the results showed that hypoxia exposure promoted the angiogenesis occurrence by immunohistochemical analysis of vascular endothelial growth factor receptor 2 (VEGFR2). Secondly, the concentration of vasodilation factor increased and it's activity was elevated during 8 h exposure, such as nitric oxide (NO) and nitric oxide synthase (NOS) (p < 0.05). Thirdly, hypoxia exposure promoted angiogenesis through up-regulation the expression of matrix metalloproteinase 2 (MMP-2), jagged, protein kinase B (AKT), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK) at 4 h; contrarily, the expression of inhibiting angiogenesis genes presented up-regulated at 8 h (p < 0.05), such as matrix metalloproteinase inhibitor-2 (TIMP-2), matrix metalloproteinase inhibitor-3 (TIMP-3). Finally, the genes and proteins that regulate angiogenesis presented obvious chronological order. Parts of them promoted the budding and extension of blood vessels were up-regulated during 4 h-8 h (p < 0.05), such as vascular endothelial growth factor a (VEGFA), VEGFR2, monocarboxylic acid transporter 1 (MCT1), CD147, prolyl hydroxylase (PHD), nuclear factor kappa-B (NF-κB); other part of them promoted blood vessel maturation were highly expressed during 12 h-24 h (p < 0.05), such as angiogenin-1 (Ang-1) and angiogenin-2 (Ang-2). In short, acute hypoxia can promote the liver angiogenesis of largemouth bass by HIF - dependent pathway.

Metabolic response provides insights into the mechanism of adaption to hypoxia in largemouth bass (Micropterus salmoides) under intermittent hypoxic conditions.

In metabolism, molecular oxygen is a necessary substrate. Oxygen imbalances are linked to a variety of circumstances in the organism's homeostasis. Recently, the positive effects of hypoxia treatment in improving exercise ability and hypoxia tolerance have become a research focus. We explored the effects of intermittent hypoxia exposure (IHE, for one hour or three hours per day) on the hypoxia tolerance of largemouth bass in this study. The results showed that (1) IHE significantly reduced the LOEcrit (the critical O2 tension for loss of equilibrium) value of largemouth bass, indicating that its hypoxia tolerance was enhanced. (2) The level of oxidative stress in the liver decreased in the HH3 group (exposed to a hypoxic condition for 3 h per day) compared to HH1 group (exposed to a hypoxic condition for 1 h per day). (3) IHE reduced the content of lactic acid and enhanced the process of gluconeogenesis in the liver. (4) Importantly, lipid mobilization and fatty acid oxidation in the liver of largemouth bass were significantly enhanced during IHE. In short, the results of this study indicate that IHE can improve hypoxia tolerance by regulating the energy metabolism of largemouth bass.

Chromosome-level genome assembly of largemouth bass (Micropterus salmoides) using PacBio and Hi-C technologies.

The largemouth bass (Micropterus salmoides) has become a cosmopolitan species due to its widespread introduction as game or domesticated fish. Here a high-quality chromosome-level reference genome of M. salmoides was produced by combining Illumina paired-end sequencing, PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. Ultimately, the genome was assembled into 844.88 Mb with a contig N50 of 15.68 Mb and scaffold N50 length of 35.77 Mb. About 99.9% assembly genome sequences (844.00 Mb) could be anchored to 23 chromosomes, and 98.03% assembly genome sequences could be ordered and directed. The genome contained 38.19% repeat sequences and 2693 noncoding RNAs. A total of 26,370 protein-coding genes from 3415 gene families were predicted, of which 97.69% were functionally annotated. The high-quality genome assembly will be a fundamental resource to study and understand how M. salmoides adapt to novel and changing environments around the world, and also be expected to contribute to the genetic breeding and other research.

Dynamic Transcriptomic Profiling During Liver Development in Schizothorax Prenanti.

Liver is an important organ for glucose and lipid metabolism, immunity, and detoxification in fish. However, the gene regulatory network of postnatal liver development still remains unknown in teleost fish. In this study, we performed transcriptome analysis on the liver of S. prenanti at three stages. A total of 1692 differentially expressed genes (DGEs) were identified across three liver developmental stages. The oil red O staining and PAS staining revealed that the lipid content of liver was increased and the glycogen content of liver was decreased during liver development. The fatty acids biosynthesis related genes were upregulated in adult and young stages compared with juvenile stage, while lipid degradation related genes were downregulated. The genes related to glycolysis, gluconeogenesis and glycogenolysis were upregulated in juvenile or young stages compared with adult stage. Further pathway analysis indicated that the CYP450 pathway, cell cycle and amino acid metabolic pathway were induced in the process of liver maturation. Our study presents the gene expression pattern in different liver development stages of S. prenanti and may guide future studies on metabolism of S. prenanti liver.

Yinchenhao Decoction ameliorates the high-carbohydrate diet induced suppression of immune response in largemouth bass (Micropterus salmoides).

Yinchenhao Decoction (YD), a Chinese herbal medicine, has been traditionally used for treatment of metabolic liver diseases. A 10-week feeding trail was carried out to examine the effects of YD supplementation in a high carbohydrate diet (HCD) on liver histopathology, immune response, disease resistance, and expression of genes associated with endoplasmic reticulum stress, autophagy, apoptosis, necroptosis and inflammation in juvenile largemouth. A diet containing 9% carbohydrate was used as a low carbohydrate diet (LCD), and a HCD was formulated to contain 18% carbohydrate and supplemented with 0, 0.5, 1, 2 or 4% YD (HCD, HCD+0.5YD, HCD+1YD, HCD+2YD and HCD+4YD). Triplicate groups of fish (5.6 ± 0.2 g) were feed the test diets to visual satiety for 10 weeks. The highest survival rate after Nocardia seriolae challenge was recorded for the HCD+4YD group. YD application led to reduced ACP, AKP, AST and ALT activities. HCD-induced cells swelling, ruptured cell membrane, migrated nuclei and increasing inflammatory cells in hepatocytes were mitigated by YD addition. Moreover, YD decreased the expressions of pro-inflammation genes (TNF-α, IL-1ß, IL-8, hepcidin1, NF-κB, COX2, CD80 and CD83) and increased the mRNA levels of anti-inflammation genes (IL-10 and IKBα). The mode of liver cell death was preferably changed to programed apoptosis rather than uncontrolled necroptosis by application of YD in HCD. Furthermore, the expression of UPR genes (IRE1, Eif2α, ATF6, XBP1 and GRP78/Bip) and autophagy genes (LC3-2, BNIP3 and P62) was increased by YD supplementation. In summary, our results demonstrated that YD addition in HCD enhances UPR, autophagy and programed apoptosis maintaining the homeostasis, and decreases uncontrolled necroptosis and inflammation, ultimately leading to improved immune response in largemouth bass.

Molecular and functional characterization of the retinol-binding protein 4 (RBP4) in hepatocytes of Schizothorax prenanti in response to palmitic acid.

Retinol-binding protein 4 (RBP4) protein is a kind of adipokines synthesized and secreted by the liver, which has been verified to play important roles in liver metabolism and energy homeostasis. However, the effects of RBP4 on hepatic lipid accumulation are still elusive in fish. In the present study, we cloned and characterized the RBP4 gene in Schizothorax prenanti (S. prenanti). RBP4 gene was specifically expressed in the liver and abdominal adipose tissue. Palmitic acid (PA; 400 µM) can significantly increase lipid deposition in primary hepatocytes after 12 h of treatment. Furthermore, RBP4 knockdown can relieve the excessive lipid deposition and endoplasmic reticulum stress in the hepatocytes caused by PA. The inhibition of RBP4 abolished the ability of PA to induce the expression of genes involved in lipogenesis and endoplasmic reticulum stress. These results demonstrate that RBP4 inhibition attenuated PA-induced lipid deposition and endoplasmic reticulum stress in hepatocytes of S. prenanti. This study could contribute to improve the understanding of RBP4 functions in the PA-induced lipid deposition in hepatocytes of fish.

Comprehensive analysis of full-length transcriptomes of Schizothorax prenanti by single-molecule long-read sequencing.

Schizothorax prenanti (S. prenanti) is one of the most important aquaculture species in the southwest of China. However, information of the full-length transcripts in S. prenanti remains unknown. In this study, single-molecule real-time (SMRT) sequencing was performed to generate full-length transcriptomes of S.prenanti. In total, 23.26 Gb of clean reads were generated. A total of 312,587 circular consensus sequences (CCS) were obtained with average lengths of 2634 bp and 84.16% (270,662) of CCS were full-length non-chimeric reads. After being corrected with Illumina library sequencing, 18,005 contigs were obtained, with 17,797 (98.81%) successfully annotated in eight public databases, including 15,839 complete open reading frames (ORFs) with an average length of 1330 bp. Furthermore, a total of 4152 alternative splicing (AS) events and 250 long non-coding RNA (lncRNA) transcripts were detected. Additionally, a total of 1129 putative transcription factors (TFs) members from 56 TF families and 11,660 simple sequence repeats (SSRs) were identified. This study provided a valuable resource of full-length transcripts for further research on S. prenanti.

High carbohydrate diet induced endoplasmic reticulum stress and oxidative stress, promoted inflammation and apoptosis, impaired intestinal barrier of juvenile largemouth bass (Micropterus salmoides).

This study assessed the effects of feed carbohydrate content on intestinal physical barrier and immunity in juvenile largemouth bass (Micropterus salmoides). Triplicate groups of juvenile fish (4.1 ± 0.2 g) were fed low (LCD, 7%), medium (MCD, 12%) and high (HCD, 17%) carbohydrate diets for eight weeks. Gut histology revealed the slight infiltration of inflammatory cells and moderate loss of mucous membrane layer in HCD group. Expression of ZO1, occluding, and claudin7 genes and epidermal growth factor receptor (EGFR) gene were significantly decreased in HCD group indicating the impairment of tight junction and epithelial cell regeneration. The results showed the significant (P < 0.05) reduction of antioxidant capacity in HCD group compared to LCD. Furthermore, expression of intestinal ERS-related genes such as IRE1, Eif2α, GRP78, CHOPα and CHOPß in HCD group was significantly higher than the LCD group. In addition, HCD induced the up-regulated expression of inflammatory (IL-8, IL-1ß, TNFα and COX2) and apoptosis (TRAF2, bax, casepase3, caspase8 and casepase9) related genes in fish intestine. The data generated in this study clearly demonstrated that HCD induced ERS and oxidative stress, which promoted intestinal inflammation and apoptosis in juvenile largemouth bass.

Chronic chlorpyrifos exposure induces oxidative stress, apoptosis and immune dysfunction in largemouth bass (Micropterus salmoides).

This study was undertaken to (a) evaluate the destructive effects of chronic exposure to low-dose of chlorpyrifos (CPF) on antioxidant system and immune function in largemouth bass (Micropterus salmoides), and (b) to examine whether dietary supplementation of curcumin can mitigate the adverse effects induced by CPF contamination. The experiment consisted of three groups (with three replicates, 30 fish per replicate) which lasted for 60 days: A control group (without CPF exposure or CU application), CP group (exposed to 0.004 mg/L of CPF), and CU group (exposed to 0.004 mg/L of CPF and fed a diet containing 100 mg curcumin per kg feed). The results showed that CPF contamination leads to reduced weight gain, severe histopathological lesions, decreased activity of antioxidant enzymes and down-regulated expression of antioxidant-related genes. Moreover, CPF upregulated the expression of pro-inflammatory genes such as TNF-α, IL-8, IL-15, downregulated anti-inflammatory genes TGF-ß1, IL-10, and promoted apoptosis through overexpression of Caspase-3, Caspase-8, caspase-9 and Bax. In addition, curcumin supplementation showed significant improvement in oxidative stress, apoptosis and immune dysfunction, but the improved effect gradually weakened during the exposure last. Gas chromatography-mass spectrometry (GC-MS) analysis for accumulation of CPF in muscle supported the changes of general physiological structure, excessive apoptotic responses, abnormal antioxidant and immune system functions and posed potential human health risks to children based on target hazard quotient. These results suggested that chronic exposure to CPF can cause oxidative stress, apoptosis and immune dysfunction, and that curcumin have the potential to reduce pesticides residues in fish. This also highlights the importance of monitoring pesticides residues in aquatic products and aquaculture aquatic environments.

Comparative transcriptome profiles of large and small bodied large-scale loaches cultivated in paddy fields.

Fish culture in paddy fields is a traditional aquaculture mode, which has a long history in East Asia. Large-scale loach (Paramisgurnus dabryanus) fast growth is suitable for paddy fields aquaculture in China. The objective of this study was to identify differential expression genes (DEGs) in the brain, liver and muscle tissues between large (LG, top 5% of maximum total length) and small (SG, top 5% of minimum total length) groups using RNA-seq. In total, 150 fish were collected each week and 450 fish were collected at twelfth week from three paddy fields for all the experimental. Histological observation found that the muscle fibre diameter of LG loaches was greater than that of SG loaches. Transcriptome results revealed that the high expression genes (HEGs) in LG loaches (fold change ≥ 2, p < 0.05) were mainly concentrated in metabolic pathways, such as "Thyroid hormone signalling pathway", "Citrate cycle (TCA cycle)", "Carbon metabolism", "Fatty acid metabolism", and "Cholesterol metabolism", and the HEGs in SG loaches were enriched in the pathways related to environmental information processing such as "Cell adhesion molecules (CAMs)", "ECM- receptor interaction" and "Rap1 signalling pathway"; cellular processes such as "Tight junction", "Focal adhesion", "Phagosome" and "Adherens junction". Furthermore, IGFs gene family may play an important role in loach growth for their different expression pattern between the two groups. These findings can enhance our understanding about the molecular mechanism of different growth and development levels of loaches in paddy fields.

MiRNA-seq analysis of spleen and head kidney tissue from aquacultured largemouth bass (Micropterus salmoides) in response to Aeromonas hydrophila infection.

Recently, the same fish diseases, which have been found in pond farming, have been found in the newly tested largemouth bass (Micropterus salmoides) system. Bacterial septicemia caused by Aeromonas hydrophila occurs frequently in largemouth bass culture leading to significant economic losses. To investigate the role miRNA in the largemouth bass disease resistance, twelve (2 tissues (spleen and head kidney) × 2 experimental groups (infected and control) × three biological replicates) small RNA libraries were constructed and sequenced with miRNA-seq. A total of 26 differentially expressed miRNAs, 8 upregulated and 18 downregulated, were identified in the spleen, and 19 differentially expressed miRNAs, 9 upregulated and 10 downregulated, were identified in head kidney (fold change ≥ 2 or ≤ 0.5 and P ≤ 0.05). The differentially expressed miRNAs with the largest fold change were selected for target gene prediction using GO and KEGG analysis. Six miRNAs in the spleen and 5 miRNAs in the head kidney were selected. Analysis showed that, of all the immune and metabolic pathways, the FoxO signaling pathway was enriched in both the spleen and head kidney groups. Common target genes of the pathway included AMP-activated catalytic subunit alpha 1 (prkaa1), phosphatidylinositol 3-kinase (pik3r3b), serine/threonine-protein kinase (plk2), and forkhead box protein G1 (foxg1a). MiRNAs (such as miR-126-5P, miR-126-3P) are involved in immune response and cell cycle functions as they regulate targeted genes in the FoxO pathway. These results will enhance our understanding of the molecular mechanisms underlying immune responses to bacterial septicemia and facilitate molecular-assisted selection of resistant strains of largemouth bass.

Rice-shrimp culture: a better intestinal microbiota, immune enzymatic activities, and muscle relish of crayfish (Procambarus clarkii) in Sichuan Province.

Intestinal microbiota play an important role in the intestinal immunity and nutrient absorption, even muscle nutritional components, and the composition and function were affected by environment. In this study, the intestinal microbiota and immune enzyme, nutritional flavor of muscle of crayfish in rice field, and pond cultivation model were compared in summer and autumn. The results of Shannon diversity and Chao 1 index of intestinal microbiota based on 16S sequencing analysis showed that the diversity and abundance in autumn were higher than in summer. And the diversity and abundance of intestinal microbiota of different model in the same season were different. Four dominant phyla (relative abundance > 5% at least in one sample) of the intestinal microbiota were Bacteroidetes, Firmicutes, Proteobacteria, and Tenericutes. From summer to autumn, the intestinal immune enzyme activity of crayfish in both models showed a decreasing trend. In summer, the activity of catalase and alkaline phosphatase of crayfish cultured in the pond was significantly higher than that in rice field (P < 0.05). In autumn, the activity of catalase and lysozyme of crayfish cultured in rice field was significantly higher than that in pond (P < 0.05). The contents of umami and sweetish amino acids of muscle were higher in rice field than in pond, and the percentage of glutamic acid and alanine was significantly higher in rice field than in pond (P < 0.05). Thus, rice field model can make crayfish a more stable intestinal environment and a better intestinal immune enzyme activity and muscular flavor. Key points • The intestinal microbiota of crayfish in rice field had tended to stabilize from summer to autumn. • The crayfish had better nutrient absorption and stronger immune abilities in the rice field. • The crayfish cultured in rice field had higher overall umami concentration than in pond.