Epizootic ulcerative syndrome causes cutaneous dysbacteriosis in hybrid snakehead (Channa maculata♀ × Channa argus♂)

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

Research on the influence of the nature of the weathered bedrock zone on the roof water bursting and sand bursting: taking Zhaogu No. 1 Mine as an example

Based on Zhaogu No. 1 Mine’s characters that are the overlying thick alluvium, multi-aquifers (groups) and thin bedrock, the water pressure of the gravel aquifer under the alluvial layer reaches 4.0 MPa, defined a high-pressure aquifer. To determine the influence of bedrock properties on roof water inrush and sand bursting, and ensure the normal mining around the thin bedrock area under groups, there were tests, point loading, dry saturated water absorption rate and indoor disintegration, of bedrock samples taken from hydrological survey holes to determine those properties and influence on retaining sand-proof pillars by analyzing the variation curves of various indexes of them with depth. The experiments’ results showed that the weathering depth of bedrock exceeds 20 m; the dry saturated water absorption rate of mudstone in the vertical depth ranging of 0−6.5 m from the bottom interface of the alluvial layer is greater than 15%. The mudstone exposed to water features muddy disintegration, broken rock fragments and mud blocks, which means it is good water-proof performance of effective bridging mining cracks and a protective layer for waterproof coal pillars; as the strength of weathered mudstone below the alluvial layer 0 to 11.4 m is lower than it of the fine gravel aquifer in the lower that of 4.0 MPa, the sand control coal pillar’s protective layer that is greater more than 11.4 m is cannot be entirely composed of weathered mudstone; due to strong resistance to disintegration and lower dry saturated water absorption rate of sandstone, the protective layer cannot be entirely composed of weathered sandstone. The compressive strength of weathered sandstone, when it is higher than 4.0 MPa, can effectively resist the overlying water head pressure

Role of nuclear pregnane X receptor in Cu-induced lipid metabolism and xenobiotic responses in largemouth bass (Micropterus salmoides)

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

Nitrification and denitrification processes in a zero-water exchange aquaculture system: characteristics of the microbial community and potential rates

​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

Influences of Trypsin Pretreatment on the Structures, Composition, and Functional Characteristics of Skin Gelatin of Tilapia, Grass Carp, and Sea Perch

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

Value-Added Carp Products: Multi-Class Evaluation of Crisp Grass Carp by Machine Learning-Based Analysis of Blood Indexes

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 (<9 N), and the remaining 252 samples were used for the analysis of seven blood indexes including hydrogen peroxide (H2O2), 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

Comparative genome-wide methylation analysis reveals epigenetic regulation of muscle development in grass carp (Ctenopharyngodon idellus) fed with whole faba bean

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

Investigation of the Performance of Solid Phase Denitrification Under Different Hydraulic Retention Times and Influent Nitrate Concentration Using Banana Stalk as a Carbon Source

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

Combined effects of dietary faba bean water extract and vitamin K3 on growth performance, textural quality, intestinal characteristics, oxidative and immune responses in grass carp

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