Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation.

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

From growth promotion to intestinal inflammation alleviation: Unraveling the potential role of Lactobacillus rhamnosus GCC-3 in juvenile grass carp (Ctenopharyngodon idella).

Lactobacillus rhamnosus is a probiotic, which not only promotes the growth of animals, but also has anti-inflammatory effects. However, the mechanism by which Lactobacillus rhamnosus regulates intestinal immunity is not well comprehended. Hence, the study aimed to research how Lactobacillus rhamnosus affects the intestinal immunity using juvenile grass carp (Ctenopharyngodon idella) as a model. We selected 1800 juvenile grass carp for testing. They were divided into six treatments and fed with six gradients of Lactobacillus rhamnosus GCC-3 (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 g/kg) for 70 days. Enteritis was subsequently induced with dextroside sodium sulfate. Results indicated that dietary Lactobacillus rhamnosus GCC-3 addition improved growth performance. Meanwhile, appropriate levels of Lactobacillus rhamnosus GCC-3 alleviated excessive inflammatory response by down-regulating the expression of TLR4 and NOD receptors, up-regulating the expression of TOR, and then down-regulating the expression of NF-κB. Additionally, appropriate Lactobacillus rhamnosus GCC-3 improved intestinal immunity by reducing pyroptosis triggered by NLRP3 inflammasome and mediated by GSDME. Furthermore, 16 S rRNA sequencing showing appropriate levels of Lactobacillus rhamnosus GCC-3 increased Lactobacillus and Bifidobacterium abundance and decreased Aeromonas abundance. These results suggest that Lactobacillus rhamnosus GCC-3 can alleviate intestinal inflammation through down-regulating NF-κB and up-regulating TOR signaling pathways, as well as by inhibiting pyroptosis.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Emerging role of vitamin D3 in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella).

Bacterial pathogens destroy the structural integrity of functional organs in fish, leading to severe challenges in the aquaculture industry. Vitamin D3 (VD3) prevents bacterial infections and strengthens immune system function via vitamin D receptor (VDR). However, the correlation between VD3/VDR and the structural integrity of functional organs remains unclarified. This study aimed to investigate the influence of VD3 supplementation on histological characteristics, apoptosis, and tight junction characteristics in fish intestine during pathogen infection. A total of 540 healthy grass carp (257.24 ± 0.63 g) were fed different levels of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) for 70 d. Subsequently, fish were challenged with Aeromonas hydrophila, a pathogen that causes intestinal inflammation. Our present study demonstrated that optimal supplementation with VD3 (1) alleviated intestinal structural damage, and inhibited oxidative damage by reducing levels of oxidative stress biomarkers; (2) attenuated excessive apoptosis-related death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling (P < 0.05); (3) enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling (P < 0.05); and (4) elevated VDR isoform expression in fish intestine (P < 0.05). Overall, the results demonstrated that VD3 alleviates oxidative injury, apoptosis, and the destruction of tight junction protein under pathogenic infection, thereby strengthening pathogen defenses in the intestine. This finding supports the rationale for VD3 intervention as an essential practice in sustainable aquaculture.

In vitro post-ovulatory oocyte ageing in grass carp Ctenopharyngodon idella affects H4K12 acetylation pattern and histone acetyltransferase activity.

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

An emerging role of arecoline on growth performance, intestinal digestion and absorption capacities and intestinal structural integrity of adult grass carp (Ctenopharyngodon idella).

Arecoline is an alkaloid with important pharmacological effects in the plant areca nut, which has been demonstrated to be an agonist of muscarinic receptors (M receptor). This study explored the influences of dietary arecoline on growth performance, intestinal digestion and absorption abilities, antioxidant capacity, and the apical junction complex (AJC) of adult grass carp (Ctenopharyngodon idella). Adult grass carp (608 to 1512 g) were fed at 6 graded levels of dietary arecoline (0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg/kg diet) for 9 weeks. The results suggested that appropriate dietary supplementation of arecoline (1.0 mg/kg) increased growth parameters and intestinal growth in adult grass carp (P < 0.05), enhanced digestion and absorption capacities (P < 0.05), up-regulated muscarinic receptor 3 (M3) mRNA level (P < 0.05), increased the content of neuropeptide fish substance P (P < 0.05), improved antioxidant capacity by activating the Keap1a/Nrf2 signaling pathway (P < 0.05), reduced intestinal mucosal permeability (P < 0.05), and increased mRNA levels of tight junction (TJ) and adherent junction AJ-related proteins in fish by inhibiting the RhoA/ROCK signaling pathway (RhoA/ROCK/MLCK/NMII) (P < 0.05). In addition, the appropriate arecoline supplementation for adult grass carp was determined to be 1.20, 1.21, 1.07, and 1.19 mg/kg based on percentage weight gain, lipase activity, serum diamine oxidase, and protein carbonyl, respectively. Overall, to the best of our knowledge, we investigated for the first time the effects and possible mechanisms of dietary arecoline on intestinal digestive and absorptive capacities and structural integrity in fish and evaluated the appropriate level of supplementation.

Re-identification and characterization of grass carp Ctenopharyngodon idella TLR20.

Toll-like receptors (TLRs) play a crucial role in the recognition of microbial-associated molecular patterns in the innate immune system. Fish TLRs have undergone significant gene expansion to adapt to complex aquatic environments. Among them, TLR20 from the TLR11 family actively responds to viral and bacterial invasions. Previous studies have reported two TLR20s in grass carp (Ctenopharyngodon idella), and in this study, we revised this conclusion. Based on the latest grass carp genome, we identified a new TLR20 member. These three TLR20s are arranged in tandem on chromosome 9, indicating that they are generated by gene duplication events. They were renamed CiTLR20.1 to CiTLR20.3 based on their chromosomal positions. The CiTLR20s in C. idella exhibit higher similarities with those in Danio rerio, Cyprinus carpio, and Megalobrama amblycephala, and lower similarities with those in other distantly related fish species. Selective pressure analysis revealed low conservation and negative evolution of TLR20s during evolution. The 3D structures of the three TLR20s showed significant differences, reflecting functional variations and different downstream adaptor molecule recruitment. Transcriptome data revealed tissue distribution differences of TLR20s, with TLR20.1 showing relatively low expression levels in all the tissues, while TLR20.2 and TLR20.3 showed higher expression in the head kidney, spleen, and gill. Additionally, TLR20.2 and TLR20.3 actively responded to GCRV-II infection, with higher upregulation of TLR20.2 in response to Aeromonas hydrophila challenge. In conclusion, this study corrected the number of grass carp TLR20 members and analyzed TLR20 from an evolutionary and structural perspective, exploring its role in antiviral and antibacterial defense. This study provides reference for future research on fish TLR20.

Structural comparison and expression function analysis of BF/C2 in Ctenopharyngodon idella and Squaliobarbus curriculus.

Ctenopharyngodon idella and Squaliobarbus curriculus, members of the Cyprinidae family and Yaroideae subfamily, have shown different levels of resistance to grass carp reo virus (GCRV), with S. curriculus exhibiting higher resilience. In the pursuit to explore the distinctions in the structural and expression traits of BF/C2 (A,B) between the two species, we conducted an analysis involving the cloning and examination of various coding sequences (CDS). We successfully cloned the CDS of ci-BF/C2A and ci-BF/C2B from C. idella, which spanned 2259 bp and 2514 bp respectively, encoding 752 and 837 amino acids. Similarly, the CDS of sc-BF/C2A and sc-BF/C2B from S. curriculus were cloned, featuring lengths of 1353 bp and 2517 bp and encoding 450 and 838 amino acids, respectively. A chromosome collinearity assessment revealed that ci-BF/C2A demonstrated collinearity with sc-BF/C2A, a finding not replicated with ci-BF/C2B and sc-BF/C2B. Delving into gene structure, we discerned that ci-BF/C2A harbored a greater number of Tryp_SPc domains compared to sc-BF/C2A. Following this, we engineered and purified six prokaryotic recombinant proteins: CI-BF/C2A, CI-BF/C2A1 (a variant resulting from the deletion of the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2A2 (representing the Tryp_SPc domain of CI-BF/C2A), CI-BF/C2B, SC-BF/C2A, and SC-BF/C2B. Through serum co-incubation tests with these recombinant proteins, we established the activation of the complement marker C3 in each case. Utilizing fluorescence quantitative expression analysis, we observed ubiquitous expression of ci-BF/C2A and ci-BF/C2B across all grass carp tissues, predominantly in the liver. This pattern mirrored in S. curriculus, where sc-BF/C2A was highly expressed in the gills, and sc-BF/C2B manifested notably in the liver. Kidney cell infection experiments on both species revealed enhanced resistance to GCRV post-incubation with the recombinant proteins. Notably, cells treated with SC-BF/C2 (A, B) exhibited pronounced resilience compared to those treated with CI-BF/C2 (A, B, A1, A2). However, cells incubated with CI-BF/C2A1 and CI-BF/C2A2 showed strengthen resistance relative to cells treated with CI-BF/C2A and CI-BF/C2B. In GCRV infection trials on grass carp, ci-BF/C2A and ci-BF/C2B expressions reached a zenith on the seventh day post-infection, highlighting a distinctive functional mode in immune defense against GCRV infection orchestrated by BF/C2. The empirical data underscores the pivotal role of the Tryp_SPc domain in immune responses to GCRV infection, pinpointing its influence on ci-BF/C2A expression. Conclusively, this investigation provides a foundational understanding of the unique immune function characteristics of BF/C2 in grass carp, paving the way for further scholarly exploration in this realm.

A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella).

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO4·5H2O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-ß1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 µm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO4·5H2O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

VP4/VP56/VP35 Virus-like Particles Effectively Protect Grass Carp (Ctenopharyngodon idella) against GCRV-II Infection.

Grass carp reovirus (GCRV) seriously threatens the grass carp (Ctenopharyngodon idella) industry. Prophylactic GCRV vaccines prepared by virus-like particle (VLP) assembly biotechnology can improve effectiveness and safety. The highly immunogenic candidate antigens of GCRV vaccines that have been generally considered are the outer capsid proteins VP4, VP56, and VP35. In this study, VP4, VP56, and VP35 were expressed in an Escherichia coli expression system and a Pichia pastoris expression system. The successful assembly of uniform, stable, and non-toxic VP4/VP56/VP35 VLPs was confirmed through various assays. After vaccination and GCRV infection, the survival rate in the VLPs + adjuvant Astragalus polysaccharide (APS) group was the highest (62%), 40% higher than that in control group (22%). Through the antibody levels, tissue viral load, and antioxidant immunity assays, the P. pastoris VLP vaccine effectively improved IgM levels, alleviated tissue virus load, and regulated antioxidant immune-related indicators. The treatment with P. pastoris VLPs enhanced the mRNA expression of important immune-related genes in the head kidney, as measured by qRT-PCR assay. Upon hematoxylin-eosin staining examination, relatively reduced tissue pathological damage was observed in the VLPs + APS group. The novel vaccine using P. pastoris VLPs as an effective green biological agent provides a prospective strategy for the control of fish viral diseases.

Variations in the Behaviour, Survival, Haematology, and Biochemistry of Ctenopharyngodon idella (Grass Carp) After Exposure to Commercial Grade Atrazine.

The current study assessed the harmful effects of Atrazine (ATZ) herbicide on haematology and biochemistry of the freshwater fish Ctenopharyngodon idella, a commercially significant fish in Pakistan. C. idella (13 ± 8.4 cm; 132 ± 5.6 g) was exposed to graded levels of ATZ, and its 96-hour LC50 value at 25°C was calculated to be 150.5 µl/L. After exposure to ATZ, fish displayed rapid movements, a loss of balance in position and equilibrium, anxious swimming patterns, colour changes, and increased mucous production. The MCHC, MCH, RBCs, and Hb in C. idella decreased significantly (P < 0.05), whereas the MCV, Ht, and WBCs were significantly increased (P < 0.05). At different time intervals (24, 48, 72, and 96 h) following ATZ administration (50, 100, 150, and 200 µl/L), biochemical analysis significantly decreased (P < 0.05) triglyceride, total protein, cholesterol, and albumin levels, whereas glucose levels significantly increased (P < 0.05). We concluded that ATZ is toxic to C. idella, altering their haematology and blood biochemistry even after only a brief exposure.

Effects of Replacing Soybean Meal Protein with Chlorella vulgaris Powder on the Growth and Intestinal Health of Grass Carp (Ctenopharyngodon idella).

Chlorella vulgaris (C. vulgaris) powder is a novel non-grain single-cell protein with enormous potential to be a protein source. However, it is poorly studied in aquatic animals. The purpose of the present study was to explore the optimum replacement ratio of C. vulgaris powder and the influence of the substitution of soybean meal with C. vulgaris on grass carp (Ctenopharyngodon idella) in terms of growth performance, intestinal integrity and the microbial community. Five isonitrogenous and isolipidic diets were formulated by replacing 0% (SM, containing 30% soybean meal), 25% (X25), 50% (X50), 75% (X75) and 100% (X100) soybean meal with C. vulgaris. The feeding trial period lasted 8 weeks. At the end of the experimental trial, the X50 group showed higher FW, WGR and PER than the SM group (p < 0.05). The feed conversion ratio (FCR) of the X50 group was significantly lower than that of the SM group (p < 0.05). The X50 group showed the highest value of the goblet cell number, intestinal amylase and trypsin activities when compared with the SM group (p < 0.05). Replacing 50% soybean meal with C. vulgaris improved the intestinal barrier integrity, as evidenced by upregulating zo-1, zo-2 and occluding transcript (p < 0.05), and alleviated oxidative stress by an increased SOD enzymatic activity and transcript level, probably mediated through the Nrf2-keap1 signaling pathway (p < 0.05). Meanwhile, the X50 group enhanced intestinal immunity, as manifested by increased ACP and LZM activities (p < 0.05), and downregulated the tlr-4, tlr-7, tlr-8 and il-6 through the tlr pathway (p < 0.05). The functionally predicting pathways related to the nitrate respiration and nitrogen respiration were observably activated in the X50 group (p < 0.05). The X50 group improved the biological barrier, as manifested by increased Firmicutes and Rhodobacter (p < 0.05). In conclusion, dietary C. vulgaris powder could promote the growth performance of grass carp by restoring intestinal morphology, increasing digestive enzyme activities, improving antioxidant properties and immunity and optimizing the microflora structure. A C. vulgaris powder replacement of 50% soybean meal was recommended as feed for grass carp.

Type II Grass Carp Reovirus Rapidly Invades Grass Carp (Ctenopharyngodon idella) via Nostril-Olfactory System-Brain Axis, Gill, and Skin on Head.

Type II grass carp reovirus (GCRV-II) with high pathogenicity and infectivity causes severe hemorrhagic disease, which leads to extensive death in the grass carp and black carp aquaculture. However, the early invasion portal remains unclear. In this study, we explored the invasion portal, time, and pathway of GCRV-II by immersion infection in grass carp. Through the detection of the infected grass carp external body surface tissues, most of them could be detected to carry GCRV-II within 45 min except for the skin covered by scales. Further shortening the duration of infection, we proved that GCRV-II rapidly invades through the nostril (especially), gill, and skin on head at only 5 min post-immersion, rather than merely by adhesion. Subsequently, visual localization investigations of GCRV-II were conducted on the nostril, olfactory system (olfactory bulb and olfactory tract), and brain via immunofluorescence microscopy and transmission electron microscopy. We found that few viruses were located in the nostril at 5 min post-immersion infection, while a significantly increased quantity of viruses were distributed in all of the examined tissues at 45 min. Furthermore, the semi-qRT-PCR and Western blotting results of different infection times confirmed that GCRV-II invades grass carp via the nostril-olfactory system-brain axis and then viral replication unfolds. These results revealed the infection mechanism of GCRV-II in terms of the invasion portal, time, and pathway in grass carp. This study aims to understand the invasion mode of GCRV-II in grass carp, thus providing theoretical support for the prevention and control strategies of hemorrhagic disease.

Promotion of fatty acid metabolism and glucose metabolism in the muscle of sub-adult grass carp (Ctenopharyngodon idella): The role of alpha-linoleic acid/linoleic acid (ALA/LNA) ratios.

The n6/n3 ratios improved meat quality of terrestrial animals, but alpha-linolenic acid/linoleic acid (ALA/LNA) ratios were rarely studied in aquatic animals. In this study, sub-adult grass carp (Ctenopharyngodon idella) were fed diets fed diets containing six varying ALA/LNA ratios (0.03, 0.47, 0.92, 1.33, 1.69, and 2.15) for 9 weeks and the total value of n3 + n6 (1.98) was kept constant for all six treatments. The results indicated optimal ALA/LNA ratio improved growth performance, changed fatty acid composition in grass carp muscle, and promoted glucose metabolism. Additionally, optimal ALA/LNA ratio improved chemical attributes by increasing crude protein and lipid contents, and technological attributes by increasing pH24h value and shear force in grass carp muscle. The signaling pathways related to fatty acid metabolism and glucose metabolism (LXRα/SREBP-1, PPARα, PPARγ, AMPK) might be responsible for these changes. Dietary optimal ALA/LNA ratio based on PWG, UFA and glucose contents was 1.03, 0.88 and 0.92, respectively.

Microbial community structure and diversity in fish-flower (mint) symbiosis.

The fish-flower symbiosis model is an eco-friendly sustainable farming technology combining plants, fish and microorganisms in a recirculating aquaculture system. However, there are few studies on the structure and diversity of microbial communities in fish intestines, culture water and plant roots during fish-flower symbiosis. Here, we cultured carp (Cyprinus carpio), crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idella) with mint (Mentha spicala L.) and extracted total genomic DNA from intestinal microorganisms, culture-water microorganisms and root microorganisms for each fish species for high-throughput sequencing of 16S rRNA genes. Analysis of microbial community structure and diversity revealed changes in abundance of microbial genera in the intestines and culture water of each fish species, including changes in the dominant taxa. Pirellula, Truepera, Aquincola, Cetobacterium and Luteolibacter were widespread in the fish intestine, culture water and mint root system. This study revealed the effects of mint feeding on the structure and diversity of microbial communities of fish, water bodies and the mint root system during fish-flower symbiosis, providing a theoretical reference for the promotion and application of fish-flower (mint) symbiosis technology and healthy fish culture technology.

LncRNA-adm2 targets adm2 via cid-miR-n3 and negatively regulates the inflammatory response in grass carp (Ctenopharyngodon idella).

Long non-coding RNAs (lncRNAs), which impact gene expression following pathogen infections, have garnered significant attention in recent years. Recent discoveries have revealed that lncRNAs play a crucial role in fish immune responses to pathogen infections. We investigated the influence of lncRNA-adm2 on the antibacterial immune response generated by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella) through the adsorption of cid-miR-n3. Furthermore, we found that cid-miR-n3 interacts with lncRNA-adm2 and targets the 3' UTR of adm2. The upregulation of lncRNA-adm2 expression led to the suppression of pro-inflammatory cytokines (il-1ß and il-6) in CIK cells, while anti-inflammatory cytokines (il-10) increased. Our research provides evidence that lncRNAs are involved in the antibacterial immune response of fish, expanding our understanding of the function of lncRNAs in teleosts.

Molecular identification and functional analysis of X-linked inhibitor of apoptosis -associated factor-1 (XAF1) in grass carp, Ctenopharyngodon idella.

X-linked inhibitor of apoptosis protein (XIAP) -associated factor 1 (XAF1) is an interferon-stimulated gene which exhibits pro-apoptosis effect. In this study, XAF1 was characterized from grass carp Ctenopharyngodon idella and its expression pattern and function were analyzed. The open reading frame (orf) of XAF1 is 789 nucleotides (nt) encoding 262 amino acids. SMART online search results showed that a C2H2-type and six C2HC-type zinc-fingers were found in XAF1, however, the XAF1 of grass carp showed high sequence identity to zebrafish (71%), low sequence identity to tetrapods (21-22%). Rt-qPCR results showed that XAF1 was constitutively expressed in all tested organs/tissues with highest expression in blood. An inductive expression of XAF1 at mRNA level was observed in peripheral blood leucocytes (PBLs) and C. idellus kidney cells (CIKs) after treatment with C. idellus recombinant interferon-γ (rIFNg). Overexpressing XAF1 in CIKs exhibited resistance against grass carp reovirus (GCRV) and more sensitivity to cisplatin. These results implied a functional homologue of XAF1 in evolution, however the mechanism may require further investigation.

6His-tatritin promotes antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp (Ctenopharyngodon idella).

Antimicrobial peptides are small, cationic, and amphiphilic peptides found in most organisms, and many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a derivative of antimicrobial peptide Tatritin, 6His-Tatritin, was designed and expressed by Pichia pastoris using a constitutive vector pGAPZαA with the promoter of pGAP. The 6His-Tatritin had a broad-spectrum antibacterial activity based on the Oxford cup method and the micro broth dilution test. In addition, to explore the role of 6His-Tatritin in vivo, grass carps (Ctenopharyngodon idellus) were infected with Aeromonas hydrophila after they were fed with 6His-Tatritin as feed additives for 28 days. The results revealed that 6His-Tatritin could significantly up-regulate the expression levels of Hepcidin, Leap-2b, Nrf-2, CuZn-SOD and LZM (P < 0.05). In addition, 6His-Tatritin could significantly reduce the mortality (P < 0.05) and the intestinal injury of grass carps infected with bacteria. The 16S sequencing analysis showed that the structure of microbial community in intestine of fish was more diversified compared with control after treatment with 6His-Tatritin. In summary, the peptide of 6His-Tatritin could promote antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp. This study provides an effective method and approach for the application of antimicrobial peptide Tatritin in aquaculture, and also provides insights into the function of antimicrobial peptides in immunity against pathogens in fish.

Longer-Term Adverse Effects of Selenate Exposures on Hematological and Serum Biochemical Variables in Air-Breathing Fish Channa punctata (Bloch, 1973) and Non-air Breathing Fish Ctenopharyngodon Idella (Cuvier, 1844): an Integrated Biomarker Response Approach.

To examine the spectrum of selenium toxicity between hardy and less hardy species of the same life stages, short-term and longer-term exposures in juvenile air-breathing fish Channa punctata (Bloch, 1973) and non-air-breathing fish Ctenopharyngodon idella (Cuvier, 1844) were assessed. Acute exposures revealed a greater 96-h median lethal concentration (LC50) for C. punctata (14.67 mg/l) compared to C. idella (7.98 mg/l). During their chronic exposure, both fishes' hemoglobin content (Hb), red blood cells (RBC), and hematocrit (HCT) markedly decreased (p < 0.05), although their clotting time (CT) significantly increased. At 96 h, immune-modulation was observed where total protein and serum globulin levels in both fishes considerably decreased (p < 0.05) compared to the first exposure at 0 days, although total glucose, triglyceride, cholesterol, and albumin levels in both fishes significantly increased (p < 0.05) at 30 days. The lower cholesterol levels in C. punctata compared to C. idella are suggestive of a disrupted cholesterol transformation pathway. The greater total protein, triglyceride, albumin, and globulin levels in C. punctata compared to C. idella are suggestive of a comparatively robust immune capacity. In essence, selenium toxicity in the wild could manifest as disrupted metabolic pathways and downregulated immune capacity for less hardy species. In general, both fish species displayed significant alterations in their hematological and biochemical responses with increased exposure duration and elevated toxicant concentrations. This comparative investigation could improve the knowledge-spectrum of selenium toxicity in the wild as well as an understanding of secondary stress responses critically evident in hematological and biochemical parameters.

Dietary cinnamaldehyde improves muscle protein content by promoting muscle fiber growth via PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling pathway in grass carp (Ctenopharyngodon idella).

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.