Search

Abstract The biodegradable polymer poly-β-hydroxybutyrate (PHB) is a promising carbon source for biological mitigation of nitrogen pollution, a significant problem in aquaculture that physical and chemical methods have not provided a comprehensive solution. Here we investigated the impact of PHB on the zero-water-change largemouth bass culture by 30- and 40-day experiments. PHB loaded into the filter circulation pump at 4 g L− 1, optimum value determined by the first experiment, significantly reduced the levels of nitrate by 99.65%, nitrite by 95.96%, and total nitrogen by 85.22% compared to the control without PHB. PHB also significantly increased denitrifying bacteria (e.g., Proteobacteria and Fusobacteria) and expression of denitrification genes (e.g., nirK and nirS) in the microbial community, improving growth and health parameters of largemouth bass. While the impact may vary in other culture systems, PHB thus demonstrated its remarkable utility in aquaculture, highlighting ecological assessment and application to larger aquaculture operations as future considerations.

The pregnane X receptor (PXR)/nuclear factor-kB (NF-kB) signaling pathway plays a critical role in regulating toxin-induced inflammation and apoptosis in mammals. Whether dietary curcumin (CUR) can prevent copper (Cu)-induced liver injury via this signaling pathway remains to be established in aquatic animals. Juvenile largemouth bass (Micropterus salmoides) were exposed to dietary Cu and CUR treatments for 8 weeks. The results showed that chronic Cu exposure induced oxidative stress, causing liver function damage and liver injury. Cu exposure stimulated inflammation by regulating nf-kb and pro-inflammatory genes such as tnfα and il-1β and promoted apoptotic signals in the liver by modulating bcl2 and casp3 mRNA levels. In addition, Pearson correlation analysis verified that inflammatory and apoptotic responses were important indicators of Cu-induced liver injury. CUR attenuated stress responses by enhancing the antioxidant system. Importantly, CUR significantly stimulated PXR mRNA and protein levels in the Cu + CUR group and suppressed NF-κB activation to inhibit the inflammatory and apoptotic signaling cascade. These results suggest that CUR may be an effective activator of PXR in teleost fishes, exerting cytoprotective effects on Cu-induced liver injury via a PXR-mediated NF-κB repression mechanism. In conclusion, this study is the first to demonstrate that CUR may act as a potent PXR ligand that exerts hepatoprotective effects against Cu-induced liver injury. The findings shed light on the specific regulatory role of the PXR/NF-κB signaling pathway in liver pathogenesis and its potential as a therapeutic target in teleost fishes.

Enteritis often limits intensive aquaculture, where bile acids (BAs), a group of steroid molecules, are used to prevent intestinal diseases. However, the functions of certain BAs are poorly understood. In this study, grass carp with enteritis (EGC) and healthy grass carp (HGC) were sampled, and the BAs' composition was analyzed. Results showed distinct differences in the BA profiles: EGC exhibited significantly elevated levels of taurodeoxycholic acid (TDCA), lithocholic acid (LCA), glycoursodeoxycholic acid (GUDCA), and glycohyocholic acid (GHCA) (P < 0.05), and decreased levels of TCDCA, taurocholic acid (TCA), and cholic acid (CA) in the intestine compared with their levels in HGC. Subsequently, five diets containing four typical BAs (CA, chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], and LCA) at the same concentration (0.1 %) were formulated to feed the juvenile grass carps (6.29 ± 0.05 g) for 8 weeks. DCA alone increased body weight, reduced the feed conversion ratio (FCR), and ameliorated histological intestinal inflammatory hallmarks. Conversely, individual supplementation with CDCA, LCA, or CA decreased body weight and increased FCR and exacerbated the histological hallmarks of enteritis. Furthermore, DCA markedly reduced the mRNA expression levels of endoplasmic reticulum stress (ERS)-related genes in the intestine, including glucose-regulated protein 78 (GPR78), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1, and protein kinase RNA-like ER kinase, as well as genes encoding proinflammatory cytokines, such as interleukin (IL)-1 beta, IL-8, and tumor necrosis factor α (TNF-α). Supplementation with CDCA, LCA, or CA promoted the expression of ERS- and proinflammatory-cytokine-related genes. Additionally, Actinobacteriota abundance negatively correlated with intestinal mRNA expression levels of TNF-α, ATF6, IL-1β, GPR78, and IL-8, and Candidatus-Arthromitus abundance positively correlated with these inflammatory markers (P < 0.05). In conclusion, our results demonstrated that BAs play differential roles in modulating the enteritis process and that DCA may have a specific anti-inflammatory role in grass carp.

Abstract Broad bean (Vicia faba L.) has received attention from the food and animal feed industries as a sustainable, inexpensive, and versatile plant‐based protein source. However, long‐term consumption of broad bean, especially unprocessed ones, can lead to hepatic lipid accumulation. To investigate how broad bean affected hepatic lipid metabolism and its underlying mechanisms, we conducted a multi‐omics analysis of crisp grass carp (Ctenopharyngodon idellus C. et V) model with hepatic steatosis after feeding with single broad bean for 120 days. The glycolysis inhibition, increased de novo lipogenesis, abnormalities in lipid metabolism, and activation of oxidative stress were the key processes involved in broad bean‐induced hepatic lipid accumulation. Disturbances in intestinal flora caused by damage to the intestinal barrier and increased lipopolysaccharide caused by broad bean promoted hepatic lipid accumulation. Reduction of antinutritional factors and balanced diet offer potential strategies to mitigate the liver damage caused by broad bean consumption. Overall, our findings provided a theoretical basis for the wide application of broad bean as animal feeds.

Habitual dietary changes have the potential to induce alterations in the host’s gut microbiota. Mandarin fish (Siniperca chuatsi), an aquatic vertebrate species with distinct feeding habits, were fed with natural feeds (NF) and artificial feeds (AF) to simulate the effects of natural and processed food consumption on host gut microbiota assemblages. The results showed that the alpha diversity index was reduced in the AF diet treatment, as lower abundance and diversity of the gut microbiota were observed, which could be attributed to the colonized microorganisms of the diet itself and the incorporation of plant-derived proteins or carbohydrates. The β-diversity analysis indicated that the two dietary treatments were associated with distinct bacterial communities. The AF diet had a significantly higher abundance of Bacteroidota and a lower abundance of Actinomycetota, Acidobacteriota, and Chloroflexota compared to the NF group. In addition, Bacteroidota was the biomarker in the gut of mandarin fish from the AF treatment, while Acidobacteriota was distinguished in the NF treatments. Additionally, the increased abundance of Bacteroidota in the AF diet group contributed to the improved fermentation and nutrient assimilation, as supported by the metabolic functional prediction and transcriptome verification. Overall, the present work used the mandarin fish as a vertebrate model to uncover the effects of habitual dietary changes on the evolution of the host microbiota, which may provide potential insights for the substitution of natural foods by processed foods in mammals.

Diets with high carbohydrate (HC) was reported to have influence on appetite and intermediary metabolism in fish. To illustrate whether betaine could improve appetite and glucose-lipid metabolism in aquatic animals, mandarin fish (Siniperca chuatsi) were fed with the HC diets with or without betaine for 8 weeks. The results suggested that betaine enhanced feed intake by regulating the hypothalamic appetite genes. The HC diet-induced downregulation of AMPK and appetite genes was also positively correlated with the decreased autophagy genes, suggesting a possible mechanism that AMPK/mTOR signaling might regulate appetite through autophagy. The HC diet remarkably elevated transcriptional levels of genes related to lipogenesis, while betaine alleviated the HC-induced hepatic lipid deposition. Additionally, betaine supplementation tended to store the energy storage as hepatic glycogen. Our findings proposed the possible mechanism for appetite regulation through autophagy via AMPK/mTOR, and demonstrated the feasibility of betaine as an aquafeed additive to regulate appetite and intermediary metabolism in fish.

In China, aquaculture is the primary source of aquatic products due to the decrease in wild fishery resources. In 2018, the total output of aquatic products in China expanded to 47.6 million tons, accounting for 58% of global aquaculture production. Intensive culture methods generally use significant quantities of feed however, approximately 75% of nitrogen in the feed is retained in aquaculture water, mainly as soluble nitrogen, such as ammonia nitrogen (NH4+-N) and nitrate (NO3–-N), owing to low feed-utilization rates during cultivation. At the same time, fishes generate a substantial amount of excreta, which will cause the increase of nitrogen compounds in water and negatively affects the quality of aquatic products. Serious problems could occur if nitrogen compounds are discharged into the environment, including the eutrophication of rivers, the deterioration of drinking water sources, and hazards to human health. Furthermore, nitrates can form potentially carcinogenic compounds, such as nitrosamines and nitrosamides, and nitrate consumption can cause methemoglobinemia in infants. The Second National Census of Pollution Sources survey showed that the total nitrogen emission from aquaculture was 99 100 tons in 2017. To protect the environment and human health, it is important to remove nitrogen from aquaculture tailwater before discharging it to the surrounding waters. Biological denitrification is considered the most promising approach since nitrate can be reduced to harmless nitrogen gas by bacteria. A sufficient carbon source is necessary during the heterotrophic denitrification process. To solve the problems mentioned above, external liquid carbon sources such as methanol, acetic acid, and glucose are added to the tailwater, but they are costly, require high-energy, and have high operating requirement. In contrast, agricultural wastes as a carbon source have exhibited significant economic advantages and high efficiency. Many aquaculture tailwater treatment systems often face variations in hydraulic retention times (HRT) and influent nitrate concentration (INC), which are caused by acute changes in tailwater characteristics and production, and HRT and INC often exert a profound effect on the treatment performance of biological treatment systems. Extensive research has confirmed that adding agricultural waste (such as corncob, woodchip and rice straw) to municipal sewage and industrial wastewater can effectively improve denitrification efficiency. However, the effect of using agricultural waste as denitrifying carbon source to treat aquaculture tail water remains unclear. Banana stalk (BS), a typical agricultural waste product, is used as a denitrifying carbon source for the first time in this study. The study investigated the effects of HRT and INC on the denitrification performance of BS, and provided a theoretical basis for the application of agricultural waste in aquaculture tailwater treatment. In this study, using BS as a carbon source and a towel as biological carrier, the performance of solid-phase denitrification under dynamic flow conditions was studied by using a 1-D column experiment. In the HRT optimization experiment, INC was maintained at 50 mg/L and operated under four HRTs (16 h, 20 h, 24 h and 28 h) for 14 days. The effluent NO3–-N, nitrite (NO2–-N), NH4+-N, Total nitrogen (TN), Total phosphorus (TP), and chemical oxygen demand (COD) removal efficiency were measured every 2 days. The optimal HRT of BS-DR (banana stalk-denitrification reactor) was optimized by one-way ANOVA analysis. Then, based on the optimization of HRT, the reactor was operated for 14 days under different INC (75 mg/L, 100 mg/L, and 125 mg/L). The sampling time interval and measurement indexes were the same as those of the HRT optimization experiment. The Illumina MiSeq high-throughput sequencing method was used to sequence and analyze the two hyper-variable regions (V3-V4) of the 16S rRNA gene of bacteria in the initial and final stages of the BS-DR. The results indicated that HRT and INC are the key factors affecting the denitrification performance of BS-DR. There was no significant difference in nitrate removal efficiency when the HRT was 20 h (96.71±1.36)%, 24 h (94.57±4.73)%, and 28 h (99.41±0.64)%, but they were significantly higher than that when the HRT was 16 h (87.53±7.95)%. Therefore, the optimal HRT for BS-DR was 20 h, and no nitrite accumulation. The second set of experiments was conducted using the optimal HRT obtained from the first set of experiments. The effluent nitrate concentration (ENC) and nitrate removal rate (NRR) of BS-DR increased significantly with increase in INC (P < 0.05), and the effluent COD decreased with increase in INC, and the proper INC for BS-DR was ≤50 mg/L. It is worth noting that BS-DR could completely remove NH4+-N in both experiments. In addition, HRT significantly affects the removal efficiency of TP, but INC has little effect. According to pyrosequencing analysis, the microbial community structure of BS-DR changed after long-term operation, with the relative abundances of Proteobacteria, Bacteroidetes, Campilobacterota, and Firmicutes increasing to 31.20%, 6.67%, 3.08%, and 4.26%, respectively, ensuring the efficient operation of the reactor. On the contrary, the relative abundances of Halobacterota, Desulfobacterota, Sva0485, Chloroflexi, and Verrucomicrobiota decreased to 10.39%, 5.13%, 2.82%, 2.00%, and 1.17 %, respectively, in the reactor. In addition, at the genus level, most of the dominant bacteria at the end of reactor operation play a role in denitrification and degradation of agricultural waste, which is significantly different from that at the beginning of the reactor operation.

The effects of dietary grape seed proanthocyanidin extract (GSPE) supplementation (0, 100, 200, and 400 mg/kg) on the stress response, antioxidant status, myofiber growth, and meat quality of Nile tilapia were investigated in present study. Dietary GSPE lowered serum stress-related parameters (cortisol, glucose, Na+, and lactate) and improved the total antioxidant capability with enhanced superoxide dismutase and catalase activities and reduced levels of reactive oxygen species. Meanwhile, myoglobin content, pH, color, tenderness, the water-holding capacity were improved. Dietary GSPE also increased the contents of delicious amino acids, flavor amino acids, and total amino acids, C18:3n-3 (ALA), C20:5n-3 (EPA), n-3 poly-unsaturated fatty acids, decreasing off-flavor compounds such as aldehydes and alcohols. Moreover, GSPE supplementation improved myofiber growth and muscle protein deposition likely via the activation of the IGFs/PI3K/Akt/TOR/S6K1/4E-BP1 pathway, contributing to the enhanced growth performance of Nile tilapia (Oreochromis niloticus). Together, GSPE is a promising natural food additive that enhances various important meat quality parameters of fish, and the optimal dietary GSPE requirement for maximum growth and muscle protein deposition was approximately 280 mg/kg.

Microcystin-LR (MC-LR) is a cyanobacterial metabolite produced during cyanobacterial blooms and is toxic to aquatic animals, and the liver is the main targeted organ of MC-LR. To comprehensively understand the toxicity mechanism of chronic exposure to environmental levels of MC-LR on the liver of fish, juvenile Nile tilapia were exposed to 0 μg/L (control), 1 μg/L (M1), 3 μg/L (M3), 10 μg/L (M10), and 30 μg/L (M30) MC-LR for 60 days. Then, the liver hepatotoxicity induced by MC-LR exposure was systematically evaluated via histological and biochemical determinations, and the underlying mechanisms were explored through combining analysis of biochemical parameters, multi-omics (transcriptome and metabolome), and gene expression. The results exhibited that chronic MC-LR exposure caused slight liver minor structural damage and lipid accumulation in the M10 group, while resulting in serious histological damage and lipid accumulation in the M30 group, indicating obvious hepatotoxicity, which was confirmed by increased toxicity indexes (i.e., AST, ALT, and AKP). Transcriptomic and metabolomic analysis revealed that chronic MC-LR exposure induced extensive changes in gene expression and metabolites in six typical pathways, including oxidative stress, apoptosis, autophagy, amino acid metabolism, primary bile acid biosynthesis, and lipid metabolism. Taken together, chronic MC-LR exposure induced oxidative stress, apoptosis, and autophagy, inhibited primary bile acid biosynthesis, and caused fatty deposition in the liver of Nile tilapia.

Solid-phase denitrification shows promise for removing nitrate (NO3−-N) from water. Biological denitrification uses external carbon sources to remove nitrogen from wastewater, among which agriculture waste is considered the most promising source due to its economic and efficiency advantages. Hydraulic retention time (HRT) and influent nitrate concentration (INC) are the main factors influencing biological denitrification. This study explored the effects of HRT and INC on solid-phase denitrification using wheat husk (WH) as a carbon source. A solid-phase denitrification system with WH carbon source was constructed to explore denitrification performance with differing HRT and INC. The optimal HRT and INC of the wheat husk-denitrification reactor (WH-DR) were 32 h and 50 mg/L, respectively. Under these conditions, NO3−-N and total nitrogen removal rates were 97.37 ± 2.68% and 94.08 ± 4.01%, respectively. High-throughput sequencing revealed that the dominant phyla in the WH-DR operation were Proteobacteria, Bacteroidetes, and Campilobacterota. Among the dominant genera, Diaphorobacter (0.85%), Ideonella (0.38%), Thiobacillus (4.22%), and Sulfurifustis (0.60%) have denitrification functions; Spirochaeta (0.47%) is mainly involved in the degradation of WH; and Acidovorax (0.37%) and Azospira (0.86%) can both denitrify and degrade WH. This study determined the optimal HRT and INC for WH-DR and provides a reference for the development and application of WH as a novel, slow-release carbon source in treating aquaculture wastewater.

Faba bean water extract (FBW) and vitamin K3 (VK3) have been demonstrated to improve the muscle textural quality of fish. To better apply these two feed additives in commercial aquaculture setting, four experimental diets (control, commercial feed group; 15% FBW, 15% faba bean water extract group; 2.5% VK3, 2.5% vitamin K3 group; combined group, 15% faba bean water extract + 2.5% vitamin K3 group) were formulated to explore their combined effects of FBW and VK3 on the growth, health status, and muscle textural quality of grass carp. The growth performance, textural quality, intestinal characteristics, and oxidative and immune responses were analyzed on days 40, 80 and 120. The results showed that supplementation with higher doses of FBW and VK3 have no influence on growth-related parameters and immune parameters of grass carp. Notably, compared with the control, fish in the combined group had the highest textural qualities (hardness, chewiness and adhesiveness), followed by those in 15% FBW and 2.5% VK3 groups (P < 0.05). Also, FBW and VK3, to some extent, may lower antioxidative ability of grass carp, as illustrated by lower levels of GSH and CAT in 15% FBW, 2.5% VK3, and combined groups on day 120 (P < 0.05). In addition, enhanced lipase activity was observed in the 15% FBW group. Taken together, the combined supplementation of FBW and VK3 was demonstrated to be a more advanced option than their individual supplementation in a commercial setting owing to the resulting combined effects on both the textural quality and health status of grass carp.

The complete replacement of fresh baits (FB) by formulated artificial feeds (AF) after domestication in mandarin fish (Siniperca chuatsi) has matured in recent years, while the underlying molecular events of long-term AF diet feeding after diet shifts remained unclear. Therefore, mandarin fish, which had been successfully domesticated, were fed with the FB or AF for 180 days to explore the molecular mechanisms underlying the differences due to shifted food habit. Compared to the FB group, slight intestinal histological damage was found in the AF group of mandarin fish. An integrated analysis of miRNA-mRNA sequencing and their interaction networks was conducted between the FB and AF groups. The result showed that a total of 209 differentially expressed mRNAs (DEmRNAs) and 2 differentially expressed miRNAs (DEmiRNAs) with significance were identified. The DEmRNAs were enriched to 39 pathways including tight junction, steroid biosynthesis and protein processing in ER. Oni-Let-7a and aca-Let-7i were the two significantly identified DEmiRNAs, whose target prediction was enriched with GO terms and KEGG pathways including TGFβ signaling pathway, steroid biosynthesis, and retinol metabolism. In general, our research was the first exploration to characterize the interacted miRNA-mRNA sequencing of mandarin fish fed the FB and AF diets, which could reveal the mechanisms of long-term adaptation to new foods in organisms who subjected to a food conversion in their early life. Data Availability Statement: The dataset generated by deep-sequencing platforms was deposited in the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra). The NCBI Sequence Read Archive accession number is PRJNA897748.

Microbial communities in aquaculture ponds play a critical role in pond productivity and the success of fish production. Nevertheless, our current understanding of the functional diversity of bacterial microbiomes in grass carp pond water and sediments is fragmentary. This study utilized high-throughput sequencing technology and community level physiological profiling (CLPP) methods to investigate microbial communities and metabolic activity of bacterial found in water samples and sediments from eight typical grass carp (Ctenopharyngodon idella) culturing pond in southern China. The correlations between putative function using PICRUSt2 and actual metabolic profiles via Biolog EcoPlates was assessed. Carbohydrates (32.51 %), amino acids (23.13 %), and polymers (16.61 %) were the most dominant carbon sources utilized by the microbial community in the sediment; while carbohydrates (34.72 %), amino acids (25.49 %), and polymers (16.15 %) were also utilized most often in the water samples. In the sediment samples, Proteobacteria (24.67 %), Chloroflexi (15.30 %), and Firmicutes (13.74 %) were the most dominant bacteria phyla. Actinobacteriota (32.62 %), Cyanobacteria (25.06 %), and Proteobacteria (17.51 %) were the most dominant bacteria phyla in the water samples. Moreover, our results suggested that the nitrogen transformations bacterial genera (Bacillus, Bradyrhizobium, Brocadia, Halomonas, and Vogesella) were positively correlated with carbon source utilization of the microbial community in the Biolog EcoPlates in the sediment. Dechloromonas and Pseudomonas were significantly negatively correlated in the water samples. In addition, there was a significant correlation between predicted function and the actual metabolic characteristics of microbial communities in sediment and water samples. The results facilitate detailed perspective of the relationship between the microbial community structure and the function in grass carp ponds and also provide a reference for the selection of suitable carbon sources in aquaculture wastewater treatment technology.

​The zero-water exchange aquaculture has been identified as a promising method of farming to decrease the environment pressure of aquaculture and to increase profits. However, the ecological functions (e.g., nitrogen removal) and microbial biodiversity of the zero-water exchange pond aquaculture system are relatively understudied. In the present study, the zero-water exchange pond aquaculture system were constructed to investigated the microbial communities, sediment potential nitrification and denitrification production rates. And five functional genes (AOA amoA, AOB amoA, nirS, nosZ, and hzsB) were used to quantify the abundance of nitrifying and denitrifying microorganisms. The results showed that the sediment of the system had excellent potential nitrification-denitrification performance, with potential nitrification and denitrification rate were 149.77-1024.44 ng N g−1 h−1 and 48.32-145.01 ng N g−1 h−1, respectively. The absolute copy numbers of nitrogen functional genes and total bacterial 16S rRNA were 1.59×105-1.39×109 and 1.55×1010-2.55×1010copies g−1, respectively, with the dominant phyla, i.e., Proteobacteria, Actinobacteriota, Chloroflexi, Cyanobacteria, and Firmicutes. The relative abundances of the genera related to nitrification and denitrification, varied from 0.01% to 0.79% and from 0.01% to 15.54%, respectively. The potential nitrification rate was positively related to the sediment TOC concentration; and the potential denitrification rate had a positive correlation with sediment nitrate concentration. The genera Bacillus positively correlated with sediment NO3‐-N concentration, whereas Flavobacterium and Shewanella positively correlated with sediment NH4+-N concentration, which could be the functional bacteria for nitrogen removal. These findings may shed light on quantitative molecular mechanisms for nitrogen removal in zero-water exchange ponds, providing a sustainable solution to nitrogen pollution problem in the freshwater aquaculture ecosystems.

To investigate the impact of betaine on high-carbohydrate-diet-induced oxidative stress and endoplasmic reticulum (ER) stress, mandarin fish (Siniperca chuatsi) (23.73 ± 0.05 g) were fed with control (NC), betaine (BET), high carbohydrate (HC), and high carbohydrate + betaine (HC + BET) diets for 8 weeks. The results showed that betaine significantly promoted the growth of mandarin fish irrespective of the dietary carbohydrate levels. The HC diet induced oxidative stress, as evidenced by significantly elevated MDA levels. The HC diet significantly stimulated the mRNA levels of genes involved in ER stress (ire1, perk, atf6, xbp1, eif2α, atf4, chop), autophagy (ulk1, becn1, lc3b), and apoptosis (bax). However, betaine mitigated HC-diet-induced oxidative stress by modulating antioxidant enzymes and alleviated ER stress by regulating the mRNA of genes in the PERK-eIF2a-ATF4 pathway. Additionally, betaine significantly reduced the mRNA levels of becn1 and bax, along with the apoptosis rate, indicating a mitigating effect on autophagy and apoptosis. Overall, dietary betaine improved growth, attenuated HC-diet-induced oxidative stress and ER stress, and ultimately alleviated apoptosis in mandarin fish. These findings provide evidence for the use of betaine in aquafeeds to counter disruptive effects due to diets containing high carbohydrate levels.

Crisp grass carp (CGC), the most representative improved varieties of grass carp (GC), features higher muscle hardness after feeding faba bean (Vicia faba L.) for 90–120 days. DNA methylation, a most widely studied epigenetic modification, plays an essential role in muscle development. Previous studies have identified numerous differentially expressed genes (DEGs) between CGC and GC. However, it remains unknown if the expression levels of these DEGs are influenced by DNA methylation. In the present study, we performed a comprehensive analysis of DNA methylation profiles between CGC and GC, and identified important candidate genes related to muscle development coupled with the transcriptome sequencing data. A total of 9,318 differentially methylated genes (DMGs) corresponding to 155,760 differentially methylated regions (DMRs) were identified between the two groups under the CG context in promoter regions. Combined with the transcriptome sequencing data, 14 key genes related to muscle development were identified, eight of which (gsk3b, wnt8a, wnt11, axin2, stat1, stat2, jak2, hsp90) were involved in muscle fiber hyperplasia, six of which (tgf-β1, col1a1, col1a2, col1a3, col4a1, col18a1) were associated with collagen synthesis in crisp grass carp. The difference of methylation levels in the key genes might lead to the expression difference, further resulting in the increase of muscle hardness in crisp grass carp. Overall, this study can help further understand how faba bean modulates muscle development by the epigenetic modifications, providing novel insights into the texture quality improvement in other aquaculture fish species by nutritional programming.

Fish skin gelatin is an important functional product in the food, cosmetics, and biomedicine industries, and establishing a green and effective fish skin gelatin extraction method is an effective way to obtain high-quality gelatin and improve its production efficiency. In this study, a trypsin method was used to extract the skin gelatin of sea perch, tilapia, and grass carp, and the microstructures of skin gelatin of these three fish species were analyzed, with such functional characteristics as thermal stability, gel strength, and emulsifying properties measured. The study results show that the skin gelatin of sea perch and tilapia obtained through the trypsin method has a relatively big molecular mass, a dense network structure, and a stable trihelix conformation. In addition, the skin gelatin of these three fish species has a relatively high β-turn content in the secondary structure, good gel strength, and water absorption properties. The compositions of the collagen-associated proteins in the skin gelatins of these three fish species extracted with the trypsin method are significantly different from each other, with positive effects of decorin and biglycan on the stability of the network structure of gelatin and a certain damaging effect of metalloendopeptidase on the network structure of gelatin. The skin gelatin of tilapia has high thermal stability and good emulsifying performance. Therefore, this gelatin type has bright application prospects in such fields as food processing, cosmetics, and drug development. In contrast, the skin gelatin of grass carp has poor functional properties. Therefore, there are significant differences among the structures and functions of skin gelatin extracted from different kinds of fish through the trypsin method. This finding has provided a useful reference for the production of customized fish gelatin according to demand.

Excessive hepatic lipid accumulation is a common phenomenon in cultured fish; however, its underlying mechanisms are poorly understood. Lipid droplet (LD)-related proteins play vital roles in LD accumulation. Herein, using a zebrafish liver cell line (ZFL), we show that LD accumulation is accompanied by differential expression of seven LD-annotated genes, among which the expression of dehydrogenase/reductase (SDR family) member 3 a/b (dhrs3a/b) increased synchronously. RNAi-mediated knockdown of dhrs3a delayed LD accumulation and downregulated the mRNA expression of peroxisome proliferator-activated receptor gamma (pparg) in cells incubated with fatty acids. Notably, Dhrs3 catalyzed retinene to retinol, the content of which increased in LD-enriched cells. The addition of exogenous retinyl acetate maintained LD accumulation only in cells incubated in a lipid-rich medium. Correspondingly, exogenous retinyl acetate significantly increased pparg mRNA expression levels and altered the lipidome of the cells by increasing the phosphatidylcholine and triacylglycerol contents and decreasing the cardiolipin, phosphatidylinositol, and phosphatidylserine contents. Administration of LW6, an hypoxia-inducible factor 1α (HIF1α) inhibitor, reduced the size and number of LDs in ZFL cells and attenuated hif1αa, hif1αb, dhrs3a, and pparg mRNA expression levels. We propose that the Hif-1α/Dhrs3a pathway participates in LD accumulation in hepatocytes, which induces retinol formation and the Ppar-γ pathway.

The pregnane X receptor (PXR) is a master xenobiotic-sensing receptor in response to toxic byproducts, as well as a key regulator in intermediary lipid metabolism. Therefore, the present study was conducted to investigate the potential role of PXR in mediating the lipid dysregulation and xenobiotic responses under Cu-induced stress in largemouth bass (Micropterus salmoides). Four groups of largemouth bass (52.66 ± 0.03 g) were treated with control, Cu waterborne (9.44 μmol/L), Cu+RIF (Rifampicin, 100 mg/kg, PXR activator), and Cu+KET (Ketoconazole, 20 mg/kg, PXR inhibitor) for 48 h. Results showed that Cu exposure significantly elevated the plasma stress indicators and triggered antioxidant systems to counteract Cu-induced oxidative stress. Acute Cu exposure caused liver steatosis, as indicated by the significantly higher levels of plasma triglycerides (TG), lipid droplets, and mRNA levels of lipogenesis genes in the liver. Liver injuries were detected, as shown by hepatocyte vacuolization and severe apoptotic signals after Cu exposure. Importantly, Cu exposure significantly stimulated mRNA levels of PXR, suggesting the response of this regulator in the xenobiotic response. The pharmacological intervention of PXR by the agonist and antagonist significantly altered hepatic mRNA levels of PXR, implying that RIF and KET were effective agents of PXR in largemouth bass. Administration of RIF significantly exacerbated liver steatosis, and such alterations were dependent on the regulations on pparγ and cd36 rather than srebp1 signaling, which suggested that PXR-PPARγ might be another pathway for Cu-induced lipid deposition in fish. Whereas, KET administration showed reverse effects on lipid metabolism as indicated by the lower hepatic TG levels, suppressed mRNA levels of pparγ and cd36. Activation of PXR stimulated autophagy and inhibited apoptosis, leading to lower hepatic vacuolization; while inhibition of PXR showed higher apoptotic signals, inhibition of autophagic genes and stimulation of apoptotic genes. Taken together, PXR played a cytoprotective role in Cu-induced hepatotoxicity through regulations on autophagy and apoptosis. Overall, our data has demonstrated for the first time on the dual roles of PXR as a co-regulator in mediating xenobiotic responses and lipid metabolism in fish, which implying the potential of PXR as a therapy target for xenobiotics-induced lipid dysregulation and hepatotoxicity.

Nitrogen pollution in aquaculture wastewater can pose a significant health and environmental risk if not removed before wastewater is discharged. Biological denitrification uses external carbon sources to remove nitrogen from wastewater; however, these carbon sources are often expensive and require significant energy. In this study, we investigated how six types of agricultural waste can be used as solid carbon sources in biological denitrification. Banana stalk (BS), loofah sponge (LS), sorghum stalk (SS), sweet potato stalk (SPS), watermelon skins (WS) and wheat husk (WH) were studied to determine their capacity to release carbon and improve denitrification efficiency. The results of batch experiments showed that all six agricultural wastes had excellent carbon release capacities, with cumulative chemical oxygen demands of 37.74–535.68 mg/g. During the 168-h reaction, the carbon release process followed the second-order kinetic equation and Ritger-Peppas equation, while carbon release occurred via diffusion. The kinetic equation fitting, scanning electron microscopy, and Fourier transform infrared spectroscopy results showed that LS had the lowest cm and the maximum t1/2 values and only suffered a moderate degree of hydrolysis. It also had the lowest pollutant release rate and cumulative chemical oxygen demand, as well as the most efficient removal of total phosphorous (TP) and total nitrogen (TN). Therefore, we concluded that LS has the lowest potential risk of excess carbon release and capacity for long-lasting and stable carbon release. The WS leachate had the highest TN contents, while the SPS leachate had the highest TP content. In the 181-h denitrification reaction, all six agricultural wastes completely removed nitrate and nitrite; however, SS had the highest denitrification rate, followed by LS, WH, BS, SPS, and WS (2.16, 1.35, 1.35, 1.34, 1.34, and 1.01 mg/(L·h), respectively). The denitrification process followed a zero-order and first-order kinetic equation. These results provide theoretical guidance for effectively selecting agricultural waste as a solid carbon source and improving the denitrification efficiency of aquaculture wastewater treatment.

Largemouth bass (Micropterus salmoides) is extremely intolerant to carbohydrates in diets. To address this problem, this work studied the effects of berberine (BBR), a Chinese herbal extract with the serum glucose-lowering activity in mammals, on regulating the serum glucose and intestinal microbiota in largemouth bass. The changes in glucose metabolism-related genes and serum metabolites were also determined to explore the possible pathways and underlying mechanisms. Our results showed that BBR reduced the serum contents of total cholesterol, triglyceride and glucose, and increased intestinal farnesoid X receptor (fxr) expression in largemouth bass (P

Texture is one of the key quality attributes used in the fresh and processed fish industry to assess product quality and consumer acceptability. To improve the textural quality of tilapia, we formulated the expanded pellet diet (EPD) and pellet diet (PD), both containing faba bean (Vicia faba, FB) water extract, a previously reported potential aquafeed additive to increase flesh texture. The common diet was used as a control. After Nile tilapia (Oreochromis niloticus) were fed three kinds of experimental diet for 120 days, muscle textural quality, growth parameters, oxidative response and immune parameters were analyzed. The results showed that there was no significant difference in the growth parameters between the three groups (P > 0.05). The highest measure of textural quality (hardness and chewiness) was found for the PD group, followed by the EPD and the control (P

We explore the prospect of applying mineral element and stable isotope data in origin tracing Procambarus clarkii to establish an origin tracing system. Microwave digestion–atomic absorption spectrometry and stable isotope ratio mass spectrometry determined the contents of 14 mineral elements (Na, Mg, Al, K, Ca, Mn, Zn, Cu, Fe, Sr, Ba, As, Se and Cd) and the abundances of C and N stable isotopes in the muscle tissue of P. clarkii from Guangdong, Hunan and Hubei regions. The one-way ANOVA and Duncan multiple comparison results revealed Na, Sr, Ba, Cu, Mn, Fe, Al, Se, δ13C and δ15N varied significantly between the three regions (p < 0.05). A systematic clustering analysis revealed the stable isotopes combined with the mineral elements easily distinguished samples into the three different regions. Multivariate statistical analysis allowed us to establish a discriminant model for distinguishing P. clarkii from the three geographical regions. When stable isotopes were combined with mineral elements, the accuracy of the linear discriminant analysis of the samples from Guangdong, Hunan and Hubei were 95%, 95% and 100%, respectively. The initial overall discriminant accuracy was 96.7%, and the cross-validation discriminant accuracy was 93.3%. Principal component analysis identified three main components which were based on eleven major factors, including Cu, Ba, Cd, Mn, δ13C, δ15N, Al and Mg, resulting in a cumulative variance contribution rate of 78.77%. We established a three-dimensional coordinate system using the three principal components to create scatter diagrams with the samples from the three regions in the coordinate system. The results revealed the samples clearly differentiated into the three regions. Therefore, mineral elements combined with stable isotopes can distinguish the regional origin of P. clarkii.

Fatty liver, characterized by excessive lipid droplet (LD) accumulation in hepatocytes, is a common physiological condition in humans and aquaculture species. Lipid mobilization is an important strategy for modulating the number and size of cellular LDs. Cyclooxygenase (COX)-mediated arachidonic acid derivatives are known to improve lipid catabolism in fish; however, the specific derivatives remain unknown. In the present study, we showed that serum starvation induced LD degradation via autophagy, lipolysis, and mitochondrial energy production in zebrafish hepatocytes, accompanied by activation of the COX pathway. The cellular concentration of PGF2α, but not other prostaglandins, was significantly increased. Administration of a COX inhibitor or interference with PGF2α synthase abolished serum deprivation-induced LD suppression, LD–lysosome colocalization, and expression of autophagic genes. Additionally, exogenous PGF2α suppressed the accumulation of LDs, promoted the accumulation of lysosomes with LD and the autophagy marker protein LC3A/B, and augmented the expression of autophagic genes. Moreover, PGF2α enhanced mitochondrial accumulation and ATP production, and increased the transcript levels of β-oxidation- and mitochondrial respiratory chain-related genes. Collectively, these findings demonstrate that the COX pathway is implicated in lipid degradation induced by energy deprivation, and that PGF2α is a key molecule triggering autophagy, lipolysis, and mitochondrial development in zebrafish hepatocytes.

Faba bean (Vicia faba, FB) is known to improve the texture of fish meat but retards growth possibly by inducing hemolysis, hepatopancreas damage, and metabolic disorder. In this study, we used ultrasonic processing to isolate four FB extracts (water extract, alcohol extract, proteins and residues) and examined their beneficial and detrimental effects. These extracts were separately mixed with commercial feed and fed to grass carp (Ctenopharyngodon idellus) using whole FB and commercial feed as controls. After fish were fed one of the six experimental diets for 50 d and 100 d, we evaluated the growth and hematological parameters, activities of metabolic enzymes, hepatopancreatic histology and oxidative response, and lipid metabolism. Results showed that both whole FB and FB residues caused growth retardation and hepatopancreas damage (P < 0.05), whereas growth performance was improved in the FB water and alcohol extract groups compared to the whole FB group. Although the FB water extract negatively affected the number and morphological parameters of red blood cells (P < 0.05), the hematological damage was less pronounced than that of the whole FB group. Excessive hepatopancreatic fat accumulation was found in the whole FB, FB alcohol extract and FB residues groups. Moreover, serious hepatopancreas damages were observed in the FB residues group. These results suggest that the beneficial and detrimental components of FB were successfully separated in the four extracts, and the FB water extract would be the best choice for grass carp culture in terms of growth performance and health. The safety evaluation of the four FB extracts would facilitate further application of FB in aquatic feed.

Avoiding and mitigating the introduction of harmful effluent into the environment must be a key part of intensive industrial aquaculture development in order to minimize pollution impacts. We constructed a novel field-scale aquaculture wastewater treatment system (FAWTS) involving three-stage purification to efficiently remove nutrients from effluent wastewater. However, the mechanisms of nutrient attenuation in the FAWTS are still unclear. Since microbiota play an important role in the treatment of aquatic pollutants, we hypothesized that the different stages of FAWTS may have enriched various nutrient-metabolizing bacteria, with these promoting nutrient attenuation. We therefore tested microbial metabolic activity, microbial composition, and their metabolic potential using Biolog-GN2 microplate culture and high-throughput sequencing of prokaryotic 16S rRNA gene amplicons. Our results showed that the FAWTS displayed high removal efficiencies for chemical oxygen demand (COD, 74.4–91.2%), total nitrogen (TN, 66.9–86.8%), and total phosphorus (TP, 76.2–95.9%). Simultaneously, microbial metabolic activity for various carbon sources was significantly enhanced in FAWTS biofilms. Denitrifying and phosphorus-removing bacteria such as Rhodobacter were enriched in these biofilms, and genes participating in denitrification and the pathway from methylphosphonate to α-D-ribose-1,5-bisphosphate were increased in the biofilm communities. These results imply that the transformed multistep purification system effectively removed N, P, and COD from aquaculture wastewater by enhancing the bacterial communities involved in these processes. This suggests that contamination-free aquaculture is a feasible goal, and that microbial communities are central to pollutant removal.

Flavor and nutritional value are important qualities of freshwater fish products, but the key factors affecting these quality parameters remain unclear. In this study, four typical aquaculture modes, including the commercial feed treatment (control), faba bean treatment (FBT), grass powder treatment (GPT), and waving water treatment with commercial feed (WWT), were used to explore the regulatory effect of water quality and feed (eaten and uneaten) on the flesh flavor and nutrition in grass carp (Ctenopharyngodon idella), a freshwater fish of the largest global production. During the culture period (90 days), water quality parameters of the four modes were measured every 15 days, and the flavor quality was evaluated by volatile flavor compounds detection and electronic nose analyzer. Flesh crude protein, crude fat, free fatty acid and free amino acid profiles were also determined. The results showed that, in the late period, the FBT mode had the poorest water quality with highest concentrations of nitrite and nitrate, while the GPT mode has the best water quality among the four modes. Most flesh flavor compounds found in the flesh of the control, GPT and WWT modes were pleasant. In the FBT mode with the poorest water quality, on the other hand, we found lower flavor quality (higher contribution of fishy compounds), higher water content, and lower contents of crude protein, crude fat, free fatty acids and free amino acids, compared to the other three modes. Correlation analyses showed that nitrite and nitrate are probably key water quality factors affecting the flavor quality and nutritional values besides eaten feed and uneaten feed factors. This study can serve as an important reference for ecological regulation and feeding administration of flesh quality in freshwater aquaculture fish.

In a preliminary screening study, our laboratory isolated from the biofloc in aquaculture waters a strain of Bacillus thuringiensis, which produced highly efficient bio-flocculants. In the present study, we extracted the crude flocculants from this strain and analyzed their properties. Distribution analysis indicated that the flocculants were mainly distributed in the supernatant of the fermentation liquid. The flocculants were extracted using an ethanol extraction method, and the chemical compositions and morphology of the crude flocculants were analyzed using the Molish reaction, Fehling reaction, ninhydrin reaction, biuret reaction, phenol-sulfuric acid assay, Coomassie brilliant blue staining, ultraviolet scanning, infrared scanning and scanning electron microscopy. The carbohydrate composition of the polysaccharides in the flocculants was analyzed with thin layer chromatography. The results indicated that flocculants were solid substances with an ivory white color and their texture was loose and soft. Visualization under scanning electron microscopy revealed that their ultra-morphology consisted of small, long and fiber-like shapes. Chemical and physical analyses indicated that polysaccharides accounted for 34.5% of the components in the crude flocculants. The monosaccharides present in crude flocculants included mainly glucose, galactose and mannitose.

Aquaculture has become a primary method to produce various aquatic products, and intensive aquaculture technologies have become commercially important. To improve the efficiency of intensive aquaculture per unit area without reducing the growth rate of cultured fish, the present study explored the potential of artificial substrata in ponds. Our results showed that the concentrations of total nitrogen (TN) and total phosphorous (TP) in the ponds with different stocking densities of grass carp were lower than those in the control group in most cases. Further, the feed conversion rate of grass carp was significantly reduced by introducing these artificial substrata, and the culture density could be significantly increased without reducing the growth rates of these fish. Artificial substrata also significantly enriched specific bacteria and changed the structure of the microbiota in pond water. The relative abundance of Proteobacteria was significantly increased, and bacteria closely related to N and P cycles, such as Hyphomicrobium, Chitinimonas, Legionella, Shewanella, Roseiflexus, and Planktothrix were significantly enhanced. These results showed that the artificial substratum could increase TN and TP removal in aquaculture pond water by enriching N and P cycle-related bacteria, thus significantly increasing the specific growth rate of grass carp and significantly reducing their feed conversion rate. Finally, the stocking density of grass carp and the yield per unit area of pond could be increased without reducing the growth rate.

Cutaneous microbiota play an important role in protecting fish against pathogens. Aphanomyces infection causes epizootic ulcerative syndrome (EUS) in fish, and by perturbing the integrity of the cutaneous microbiota, increases the potential for infection by pathogenic bacteria. However, whether the composition of the cutaneous microbiota is altered in fish with EUS, and if so, which species are changed and how this might influence infected fish, is still largely unclear. Considering the importance of cutaneous microbiota in maintaining host health, we hypothesized that Aphanomyces infection significantly enhances the presence of certain bacterial pathogens in the cutaneous microbiota and causes cutaneous dysbacteriosis. To test this hypothesis, we compared the cutaneous microbiota compositions of hybrid snakehead (Channa maculata♀ × Channa argus♂) with and without Aphanomyces infection using Illumina Miseq sequencing of the 16S rRNA gene. Our results showed that the cutaneous microbiota of hybrid snakehead were significantly altered subsequent to EUS infection and that the numbers of potentially pathogenic bacteria classified into the genera Anaerosinus, Anaerovorax, Dorea, and Clostridium were significantly enhanced in the cutaneous microbiota of hybrid snakehead with EUS, whereas bacteria classified into the genera Arthrobacter, Dysgonomonas, Anoxybacillus, Bacillus, Solibacillus, Carnobacterium, Lactococcus, Streptococcus, Achromobacter, Polynucleobacter, Vogesella, and Pseudomonas were significantly reduced. These results imply that treatment for EUS should not only take into consideration the control of Aphanomyces reproduction but should also focus on regulating the cutaneous microbiota of infected fish.

Background: Although functional bacteria are widely used in aquaculture water treatment, whether they affect the indigenous microbiota and whether the impact is persistent remain unclear. Therefore, we aimed to explore the denitrification effect of Pseudomonas furukawaii ZS1 isolated from a grass carp culture pond in nitrogen-rich aquaculture water, and determine whether its effect on the microbiota structure of the aquaculture water was persistent. Methods: Three each of treatment and control groups were set up, and P. furukawaii ZS1 was added to the treatment group. The concentrations of ammonia nitrogen, nitrite, and nitrate, and the pH of each sample were measured for eight consecutive days. Changes of microbiota composition in the water were analysed via high-throughput sequencing. Results: Ammonia, nitrite, and nitrate concentrations were substantially lower in the treatment group than in the control group. There were significant differences in the microbiota structure between treatment and control groups, especially on days 2–7 after adding P. furukawaii ZS1. Furthermore, significantly enriched bacterial genera in the treatment group were initially higher in number than inhibited genera, but subsequently reverted to being lower in number. Conclusions: These results provide theoretical guidance for the effective use of P. furukawaii ZS1 to control aquaculture water.

Crisp grass carp products from China are becoming more prevalent in the worldwide fish market because muscle hardness is the primary desirable characteristic for consumer satisfaction of fish fillet products. Unfortunately, current instrumental methods to evaluate muscle hardness are expensive, time-consuming, and wasteful. This study sought to develop classification models for differentiating the muscle hardness of crisp grass carp on the basis of blood analysis. Out of the total 264 grass carp samples, 12 outliers from crisp grass carp group were removed based on muscle hardness (2O2), glucose 6-phosphate dehydrogenase (G6PD), malondialdehyde (MDA), glutathione (GSH/GSSH), red blood cells (RBC), platelet count (PLT), and lymphocytes (LY). Furthermore, six machine learning models were applied to predict the muscle hardness of grass carp based on the training (152) and testing (100) datasets obtained from the blood analysis: random forest (RF), naïve Bayes (NB), gradient boosting decision tree (GBDT), support vector machine (SVM), partial least squares regression (PLSR), and artificial neural network (ANN). The RF model exhibited the best prediction performance with a classification accuracy of 100%, specificity of 93.08%, and sensitivity of 100% for discriminating crisp grass carp muscle hardness, followed by the NB model (93.75% accuracy, 91.83% specificity, and 94% sensitivity), whereas the ANN model had the lowest prediction performance (85.42% accuracy, 81.05% specificity, and 85% sensitivity). These machine learning methods provided objective, cheap, fast, and reliable classification for in vivo crisp grass carp and also prove useful for muscle quality evaluation of other freshwater fish.

Constant consumption of broad bean (Vicia faba L.) induces intestinal inflammation and reduces growth rate in grass carp (Ctenopharyngodon idellus C. et V). However, the mechanisms underlying these effects are unclear. In mammalian models of inflammatory bowel disease (IBD), endotoxin and flagellin cause intestinal inflammation through upregulation of tumor necrosis factor (TNF)-α expression. We therefore speculated that broad bean consumption alters intestinal microbiota composition, thereby increasing the relative abundance of endotoxin-producing Gram-negative and flagellated bacteria and resulting in upregulation of TNF-α and intestinal inflammation in grass carp. We tested this hypothesis by comparing intestinal microbiota compositions of grass carp fed broad bean (GCBB), hybrid giant napier (Pennisetum sinese Roxb, GCHG), or formula feed (GCFF) by 16S rRNA gene sequencing. We also performed a histological analysis of the intestinal inner wall by scanning electron microscopy and measured intestinal wall and serum concentrations of TNF-α. Our results revealed epithelial cell damage including microvillus effacement and synechia along with increased TNF-α levels in the intestinal wall in the GCBB group as compared to the GCHG and GCFF groups. The relative abundances of Gram-negative and flagellated bacteria were also higher in the GCBB group than in the GCHG and GCFF groups; this was accompanied by upregulation of genes expressing endotoxin and flagellin in intestinal microbiota. Thus, broad bean-induced intestinal inflammation in grass carp shares features with IBD. Our findings demonstrate that the microbiome in fish is directly influenced by diet and provide a reference for deconstructing host–intestinal microbiota interactions.