Grass Carp Culture Research Articles

Evaluation of virulence of Aeromonas veronii strain GZ21-2 and development of a highly effective vaccine for grass carp with the potential for industrial application

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C–34 °C, respectively. The median lethal dose (LD50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii, incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application.

The effects of GCRV on various tissues of grass carp (Ctenopharyngodon idella) and identification of differential interferon-stimulating genes (ISGs) through muscle transcriptome analysis

Grass carp hemorrhagic disease is caused by the grass carp reovirus (GCRV). The disease spreads rapidly and has a high fatality rate, which seriously affects grass carp culture. Moreover, the molecular mechanisms underlying grass carp hemorrhagic disease remain unclear. To decipher the effects of GCRV on grass carp tissues, resistant grass carp A (GA) and susceptible grass carp B (GB) were selected through GCRV treatment, and control grass carp C (GC) was also established. The gill, liver, and muscle tissues exhibited different onset symptoms under the influence of GCRV by histological observation. We selected muscle samples with significant differences in symptoms for Illumina RNA sequencing. Analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed 3512, 3074, and 1853 differentially expressed genes between "GC vs. GB," "GC vs. GA," and "GA vs. GB," respectively. Additionally, 40 differential immune-related genes and 28 differential interferon-stimulating genes (ISGs) related to the interferon (IFN) pathway were identified. The expression of immunogene-related genes of GB and GA, such as MDA5, IL-34, NF-KB, TRIM25, SOCS3, CEBPB, and BCL2, and genes associated with the JAK-STAT signaling pathway, such as IRF4, STAT1, STAT3, JAK 1, and JAK 2, was significantly upregulated. The IFN and JAK-STAT signaling pathways were closely related to anti-GCRV infection. The transcriptome data and predicted immune genes and ISGs in this study provide novel insights into the treatment of GCRV.

Building adjustment capacity to cope with running water in cultured grass carp through flow stimulation conditions

The adaptability of cultured fish to complex flow conditions is crucial for their survival after being released into the wild. Running water in natural environments poses significant challenges for the proliferation and release of cultured fish. This study aimed to investigate the effects of flow stimulation on the adjustment capacity of cultured fish to cope with running water. The target fish were cultured grass carp. An annular flume was used to conduct tests on training and control groups. The results demonstrated an enhancement in the adjustment capacity of cultured fish following appropriate flow stimulation training. (1) The trained fish exhibited a heightened preference for low-velocity areas. (2) The trained fish displayed the ability to select a route characterized by low energy consumption, predominantly following the periphery of the low-velocity area. This suggested that an appropriate flow velocity could improve the sensitivity of training fish to water flow information, and their adjustment capacity to cope with running water improved to a certain extent. A higher adjustment capacity allowed them to process flow rate information rapidly and identify a migration strategy with lower energy consumption. This study provides a useful reference for enhancing the survival rate of grass carp through stock enhancement initiatives and contributes to the sustainability of freshwater ecosystems.

Mechanical abrasion stimulation: Altered epidermal mucus composition and microbial community in grass carp (Ctenopharyngodon idella)

In modern high-density farming, seasonal change, fishing, transportation, and other conditions easily cause mechanical abrasion of fish skin. This leads to alterations in the material balance of the epidermal mucus composition of fish, preventing normal immune function. Subsequently, the risk of disease increases. In this study, grass carp (Ctenopharyngodon idella) was used as an object to investigate immune-related enzyme activities, structural characteristics, and physiological changes of its epidermal mucous cells caused by mechanical abrasion. Furthermore, we explored the changes in body surface mucus composition and microbial community diversity and structure. Our results showed that mechanical abrasion stimulation, significantly changed the activities of immune-related enzymes, such as lysozyme (LZM), alkaline phosphatase (AKP), and acid phosphatase (ACP) were within 24 h (P < 0.05). Their levels gradually recovered after 48 h. In contrast, no significant change was observed in superoxide dismutase (SOD) levels. The number of mucous cells increased, especially those of type I and type II mucous cells in the dorsal skin, which were significantly increased (P < 0.05). Meanwhile, the stimulation changed the metabolites composition of the fish epidermal mucus in the short term, upregulating 12-OPDA, indole, quinic acid and GSH, which are beneficial to antibacterial and anti-inflammatory, and downregulating L-saccharopine, L-2-aminoadipic acid, spermine, GSSG and ribose 5-phosphate, which disrupt protein synthesis, energy production and DNA synthesis. Additionally, these metabolite changes further affect the composition of microorganism communities, rising the abundance of certain disease-associated microorganisms, such as Acidovorax, Rhizobiaceae, Aurantimicrobium, and Leifsonia, which increases the risk of disease in fish. The findings of the present study provide a deeper understanding of the role of mucus as a component of the immune system in fish and provide a scientific basis for effective grass carp culture management and disease control.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella)

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1β, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.

Microbiota composition and correlations with environmental factors in grass carp (Ctenopharyngodon idella) culture ponds in South China.

To maintain the health of aquaculture fish, it is critical to understand the composition of microorganisms in aquaculture water and sediment and the factors affecting them. This study examined the water and sediment microbiota compositions of four different types of ponds in South China that were used to culture grass carp (Ctenopharyngodon idella) of different sizes through high-throughput sequencing of the 16S rRNA gene, and analyzed their correlations with environmental factors. The results showed that ponds with cultured grass carp of different sizes exhibited significant differences in terms of water physicochemical properties and composition of water and sediment microbiota. Furthermore, the exchange of microorganisms between water and sediment microbiota was lowest in ponds with the smallest grass carp and highest in ponds with the largest grass carp. All detected environmental factors except water temperature were significantly correlated with the water microbiota, and all detected environmental factors in the sediment were correlated with sediment microbiota. Moreover, Aeromonas hydrophila and Vibrio were significantly increased in the water microbiota, especially in ponds with small juvenile grass carp, implying an increased risk of A. hydrophila and Vibrio infections in these environments. Our results provide useful information for the management of grass carp aquaculture ponds.

Strengthening and microbial regulation mechanism of Bacillus on purification device for grass carp culture wastewater

Aquaculture wastewater (AW) poses a threat to natural aquatic environments. Microecological agents are widely used to regulate and purify AW, with Bacillus being the most common. To evaluate the AW purification effect of adding Bacillus subtilis and Bacillus licheniformis to an AW treatment device, we constructed an experimental device including a small grass carp culture pond and three groups of cuboid reactors. The effects of adding the two strains to the AW treatment reactor on the AW purification effect and the microbiota compositions in the AW and packing surface biofilm were analyzed via high-throughput sequencing of the 16S rRNA gene. Our results showed that adding Bacillus bacteria to reactors improved the total nitrogen (TN) removal efficiency and reduced the chemical oxygen demand (COD). Adding both the B. subtillis and B. licheniformis preparations significantly increased the abundance of Firmicutes in the water microbiota of the reactor at the middle and end stages of the experiment. The addition of Bacillus changed the microbiota composition in the water and packing surface biofilm and significantly increased the abundance of Bacillus at the middle and later stages of the experiment. Therefore, the addition of Bacillus improved the TN removal efficiency in the AW grass carp treatment reactors and significantly reduced the COD in the AW by increasing the abundance of Bacillus and changing the microbiota composition in the system. We provide an effective way for improving the purification capacity of biofilm reactor.

Development of a rapid and sensitive reverse transcription real-time quantitative PCR assay for detection and quantification of grass carp reovirus II

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection.

Hsp90 Regulates GCRV-II Proliferation by Interacting with VP35 as Its Receptor and Chaperone.

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.

Transcriptomic Analysis of the Liver and Brain in Grass Carp (Ctenopharyngodon idella) Under Heat Stress.

Temperature is a major environmental factor that influences growth, development, metabolism, and physiological performance in fish. Grass carp (Ctenopharyngodon idella) is a highly productive fish in freshwater culture. To understand the molecular mechanism of grass carp under heat stress, we used RNA-Seq to analyze the liver and brain transcriptome of 12 libraries constructed from high-temperature (36 °C) and control (28 °C) groups. We obtained 42.49 and 42.57GB of clean data from six liver and six brain libraries, respectively, and identified 2,534 genes that were differentially expressed in liver tissue and 1622 in brain tissue (P < 0.05). According to KEGG analysis, significant differences occurred in the expression of genes involved in metabolic and immune pathways, such as the cAMP signaling pathway, apoptosis, calcium signaling pathway, lipid metabolism, and protein processing in endoplasmic reticulum and peroxisome proliferator-activated receptor signaling pathways. This study revealed that high temperature enhanced lipid metabolism, reduced fatty acid synthesis, and disrupted the immune system of grass carp. These results investigated the molecular regulation of heat stress in grass carp and provided valuable information for the healthy culture of grass carp under high temperatures.

Bacillus velezensis LG37 absorbs and utilizes ammonia nitrogen from aquaculture water and enhances the toxicity tolerance of grass carp (Ctenopharyngodon idella) to ammonia (non-ionic ammonium)

High concentrations of ammonia nitrogen lead to multi-organ damage, decreased immunity, and metabolic disorders in aquaculture animals, which cause disease outbreaks. This study mainly screened probiotic strains for good control of the concentration of ammonia nitrogen and identified strain LG37 as Bacillus velezensis according to its morphological, 16S rDNA, phylogenetic, and genomic data analysis. This LG37 strain achieved efficiently purifies aquaculture water and enhances the toxicity tolerance of grass carp to ammonia. The experimental on growth characteristics showed that LG37 exhibited a satisfactory growth trend with 10 types of sugar, 17 types of amino acids, and 6 types of organic nitrogen as the carbon or nitrogen source. Under the conditions of initial dissolved oxygen at 6±1 mg/L, the temperature at 27±2°C, and pH 7±0.3. Through wastewater treatment equipment, which degraded ammonia nitrogen and the feed protein in grass carp culture wastewater reached up to 55.5% and 73.6% within one week, respectively. Moreover, in the acute toxicity test, when feed without LG37 was fed to grass carp, the safe concentration of ammonia nitrogen (non-ionic ammonium) was 3.37 mg/L (0.2 mg/L). When feed with LG37 was fed to grass carp, the safe concentration of ammonia nitrogen (non-ionic ammonium) was 5.11 mg/L (0.31 mg/L). Compared to feed without LG37, the feed with LG37 increased the toxicity tolerance of grass carp to ammonia nitrogen (non-ionic ammonium) by 51.63% (55%).

Oral Administration of Bacillus subtilis Subunit Vaccine Significantly Enhances the Immune Protection of Grass Carp against GCRV-II Infection

Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s-CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes (TNF-α, IL-1β, IFN1 and MHC-II) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases.

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction.

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHβ and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

Effect of the Aerobic Denitrifying Bacterium Pseudomonas furukawaii ZS1 on Microbiota Compositions in Grass Carp Culture Water

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.

Effects of Bacillus methylotrophicus WM‐1 on water quality dynamics and cultural fish in grass carp culture system

Microbial agents have been widely used in the Chinese aquaculture industry due to their advantages of enhancing the immunity of aquatic animal, improving disease resistance and improving the aquaculture environment. In previous studies, we found a highly antagonistic Aeromonas hydrophila strain, Bacillus methylotrophicus WM-1, isolated from grass carp (Ctenopharyngodon idella) culture systems. The purpose of this study was to investigate the impact of WM-1 on cultured water and fish. Strain WM-1 was mixed into the feed and fed to the grass carp for 30 days, set control group (CG, 0 CFU/g feed), low concentration group (LG, 103 CFU/g feed), medium concentration group (MG, 105 CFU/g feed) and high concentration group (HG, 107 CFU/g feed). Water quality and physiological indicators of grass carp were tested. Compared with CG, the concentrations of total phosphorus (TP) and soluble reactive phosphorus (SRP) were significantly increased in the MG in the early stage (before day 18, p < 0.05), whereas decreased in the long stage. After the 12th day, treatment groups showed a significant decrease in chemical oxygen demand (COD) (p < 0.05). Total nitrogen (TN), ammonia nitrogen (), nitrite () and nitrate () had no significant difference (p > 0.05). In grass carp serum, GPT activity in MG and HG was significantly reduced, SOD activity was significantly increased, and POD in MG was significantly higher than LG and HG (p < 0.05). In the liver, the GPT activity of the LG was significantly increased (p < 0.05). Other indicators (the specific growth rate and condition factor of grass carp, serum GOT, AKP and POD activity, total liver protein content, liver SOD, POD, AKP and GPT activity, haemocyte counts) have no significant difference between CG and treatment groups (p > 0.05). The results show that WM-1 is safe for water quality and culture objects, and has the effects of improving water quality and the immunity of grass carp. The corrent results combined with WM-1 effectively inhibit A. hydrophila shown that WM-1 can be applied to aquaculture production as a probiotics.

Optimizing the immunization procedure of single-walled carbon nanotubes based vaccine against grass carp reovirus for grass carp

Hemorrhagic disease caused by grass carp reovirus (GCRV) is considered to be one of the most serious threats to grass carp, resulting in significant economic loss in grass carp culture industry. This study was aimed at optimizing the immunization program of SWCNTs-vaccine (SWCNTs-M-VP4-3) controlling infectious GCRV, determining the best immunization program for grass carp, and then providing reference for the commercial promotion and use of vaccines in aquaculture. We designed an compete experiment to optimize different parameters affecting vaccination such as immunization time, antigen concentration, and fish density when grass carp was vaccinated by bath treatment. Our results showed that the highest relative percent survival (88.33%) was found in 12 h of immunization time, 10 mg L−1 of antigen concentration, and 15 fish per liter of fish density compared to 100% mortality for control groups. And other immune responses (serum antibody production, enzyme activities, and immune genes expression) also demonstrated similar results. These results indicated that the administration protocol which induce highest immune response of the host, had the highest vaccine effect against the disease. This study lays the foundation for providing reference for the commercial promotion and use of vaccines in aquaculture.

Use of Asian watergrass as feed of grass carp

Grass carp (Ctenopharyngodon idella) was cultured in the inundated low-lying coastal wetlands using Asian watergrass (Hygroryza aristata) as feed for 6 months. The study was carried out with three treatments, each with three replications. The grass was planted during plot preparation in treatment one (Grass Planted; GP); the grass was collected those grown in an adjacent land and supplied to the fish in treatment two (Grass Supplied; GS); and the fish were fed with commercial feed in treatment three (Commercial Feed; CF). Similar sized juveniles of grass carp (51 ± 6 g) were stocked at the rate of 12,500 fish ha−1 in each treatment. Important water quality parameters were measured at a monthly interval and found within the suitable range for growth of grass carp. The survival was significantly higher in GP (92.7 ± 1.0 %), followed by GS (90.0 ± 1.7 %) and CF (90.0 ± 2.9 %). The growth parameters (weight gain and specific growth rate; SGR) of fish were found significantly higher in GP, followed by CF and GS. The highest total production was found in GP (6770 ± 424 kg ha−1), followed by CF (6733 ± 536 kg ha−1) and GS (5519 ± 483 kg ha−1). The highest net economic benefit and benefit-cost ratio were found in GP (US$ 13375.50 ha−1 and 3.32) and the lowest in CF (US$ 4958.25 ha−1 and 0.42). The results of the present study indicate that Asian watergrass can successfully be used in commercial culture of grass carp and it may significantly reduce the production cost.

Safety evaluation of four faba bean extracts used as dietary supplements in grass carp culture based on hematological indices, hepatopancreatic function and nutritional condition

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.

Intestinal microbial profiling of grass carp (Ctenopharyngodon idellus) challenged with Aeromonas hydrophila

Bacterial enteritis caused by Aeromonas hydrophila is one of the most common diseases in grass carp culture. There have been intensive reports on grass carp after A. hydrophila infection at the molecular level, but analyses of the intestinal microbiota are rare. In this study, histopathological sections were examined, and 16S rRNA sequencing was used to study the changes in the intestinal tissue and microbiota of grass carp after infection with A. hydrophila. Histological analysis indicated that the symptoms appeared at 12 h, were most severe at 6 d and 9 d, and did not recover until 15 d. Based on the community structure and diversity of the intestinal microbiota, acute fluctuations and dramatic increases occurred at 12 h, with decreases at 24–72 h, a rapid increase at 6–9 d and subsequent somewhat recovery. Four phyla (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes), especially Proteobacteria, reflected fluctuations in the intestinal microbial composition at different time points. Potential functions also showed that immune- or disease-related pathways might dominate as feedback responses to pathogen (A. hydrophila) stimulation, and the expression of most of the marker functions increased from 0 h to 12 h, decreased at 24 h–72 h, increased to a peak at 6 d or 9 d, and then again decreased at 15 d. These findings could elucidate the dynamic changes in the intestinal microbiota that occur in response to A. hydrophila infection and might shed light on the mechanism of fish intestinal pathogenesis from the perspective of the intestinal microbiota.

Comprehensive analysis of circRNA expression pattern and circRNA–mRNA–miRNA network in Ctenopharyngodon idellus kidney (CIK) cells after grass carp reovirus (GCRV) infection

Grass carp reovirus (GCRV) causes grass carp hemorrhage disease and is one of the most damaging factors in the culture of grass carp (Ctenopharyngodon idellus). Until now, the mechanisms underlying GCRV infection have been largely unknown. To explore the molecular responses to GCRV infection in C. idellus kidney (CIK) cells, we used high-throughput sequencing to investigate the circRNA, mRNA, and miRNA expression patterns and the circRNA–miRNA–mRNA network. A total of 76 circRNAs, 798 mRNAs, and 186 miRNAs were identified as differentially expressed (DE) RNAs in GCRV-infected cells compared with uninfected cells, of which 40 circRNAs, 658 mRNAs, and 146 miRNAs were upregulated, and 36 circRNAs, 140 mRNAs, and 40 miRNAs were downregulated (fold change >2, p < .05). A Gene Ontology (GO) analysis of the DE circRNA parental genes, mRNAs, and miRNA target genes was performed. The parental genes of the DE circRNAs, the DE mRNAs, and the target genes of the DE miRNAs were mainly enriched in metal-ion-binding-, immune-, and DNA-replication-related items, respectively. A KEGG pathway analysis showed that the DE circRNA parental genes, DE mRNAs, and miRNA target genes were predominantly enriched in endocytosis-, immune-, and infection-related pathways, respectively. The miRNA–target interactions were also predicted and competing regulatory networks of endogenous RNAs were constructed based on the DE RNAs. Our prediction results suggest that the circRNA-2780/cik–miRNA-7796/KAT6B, circRNA-1351/cik-miRNA–10648/B2L14 and circRNA-1591/cik-miR-8141/AMPD3 axes may be associated with GCRV infection. This study suggests that the crosstalk between circRNAs and their competing mRNAs play crucial roles in GCRV infection.