Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Multiple anomalies in wild-caught fish species Curmuca barb Hypselobarbus curmuca (Hamilton 1807) (Cyprinidae:Cypriniformes) from the Western Ghats of India.

Fish of the genus Hypselobarbus (Bleeker 1860) are widely dispersed in the rivers of the Western Ghats in India and endemic to southern Indian peninsular freshwaters. These are small- to medium-sized fishes of the family Cyprinidae. Although fish with deformed bodies or body parts are rare in natural waters, this article deals with four abnormal specimens of Hypselobarbus curmuca (Hamilton 1807) collected from the rivers Tunga, Bhadra, and Kali during 2022. The abnormalities observed in four different individuals are pughead deformity, pelvic fin deformity, pectoral fin deformity, and enlarged scales. The morphological comparison of normal individuals of Hypselobarbus curmuca (Hamilton 1807) with abnormal specimens revealed variation. Using the MT-COI gene, species identity was confirmed and the mean genetic divergence between the normal and abnormal specimens was estimated to be less than 1%.

Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Motilin, a Novel Orexigenic Factor, Involved in Feeding Regulation in Yangtze Sturgeon (Acipenser dabryanus).

Motilin is a gastrointestinal hormone that is mainly produced in the duodenum of mammals, and it is responsible for regulating appetite. However, the role and expression of motilin are poorly understood during starvation and the weaning stage, which is of great importance in the seeding cultivation of fish. In this study, the sequences of Yangtze sturgeon (Acipenser dabryanus Motilin (AdMotilin)) motilin receptor (AdMotilinR) were cloned and characterized. The results of tissue expression showed that by contrast with mammals, AdMotilin mRNA was richly expressed in the brain, whereas AdMotilinR was highly expressed in the stomach, duodenum, and brain. Weaning from a natural diet of T. Limnodrilus to commercial feed significantly promoted the expression of AdMotilin in the brain during the period from day 1 to day 10, and after re-feeding with T. Limnodrilus the change in expression of AdMotilin was partially reversed. Similarly, it was revealed that fasting increased the expression of AdMotilin in the brain (3 h, 6 h) and duodenum (3 h), and the expression of AdMotilinR in the brain (1 h) in a time-dependent manner. Furthermore, it was observed that peripheral injection of motilin-NH2 increased food intake and the filling index of the digestive tract in the Yangtze sturgeon, which was accompanied by the changes of AdMotilinR and appetite factors expression in the brain (POMC, CART, AGRP, NPY and CCK) and stomach (CCK). These results indicate that motilin acts as an indicator of nutritional status, and also serves as a novel orexigenic factor that stimulates food intake in Acipenser dabryanus. This study lays a strong foundation for the application of motilin as a biomarker in the estimation of hunger in juvenile Acipenser dabryanu during the weaning phase, and enhances the understanding of the role of motilin as a novel regulator of feeding in fish.

Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.

Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Functional characterization of Malabar grouper (Epinephelus malabaricus) interferon regulatory factor 9 involved in antiviral response.

IRF9 is a crucial component in the JAK-STAT pathway. IRF9 interacts with STAT1 and STAT2 to form IFN-I-stimulated gene factor 3 (ISGF3) in response to type I IFN stimulation, which promotes ISG transcription. However, the mechanism by which IFN signaling regulates Malabar grouper (Epinephelus malabaricus) IRF9 is still elusive. Here, we explored the nd tissue-specific mRNA distribution of the MgIRF9 gene, as well as its antiviral function in E. malabaricus. MgIRF9 encodes a protein of 438 amino acids with an open reading frame of 1317 base pairs. MgIRF9 mRNA was detected in all tissues of a healthy M. grouper, with the highest concentrations in the muscle, gills, and brain. It was significantly up-regulated by nervous necrosis virus infection and poly (I:C) stimulation. The gel mobility shift test demonstrated a high-affinity association between MgIRF9 and the promoter of zfIFN in vitro. In GK cells, grouper recombinant IFN-treated samples showed a significant response in ISGs and exhibited antiviral function. Subsequently, overexpression of MgIRF9 resulted in a considerable increase in IFN and ISGs mRNA expression (ADAR1, ADAR1-Like, and ADAR2). Co-immunoprecipitation studies demonstrated that MgIRF9 and STAT2 can interact in vivo. According to the findings, M. grouper IRF9 may play a role in how IFN signaling induces ISG gene expression in grouper species.

Deciphering dynamic interactions between spermatozoa and the ovarian microenvironment through integrated multi-omics approaches in viviparous Sebastes schlegelii.

The ovarian microenvironment plays a crucial role in ensuring the reproductive success of viviparous teleosts. However, the molecular mechanism underlying the interaction between spermatozoa and the ovarian microenvironment has remained elusive. This study aimed to contribute to a better understanding of this process in black rockfish (Sebastes schlegelii) using integrated multi-omics approaches. The results demonstrated significant upregulation of ovarian complement-related proteins and pattern recognition receptors, along with remodeling of glycans on the surface of spermatozoa at the early spermatozoa-storage stage (1 month after mating). As spermatozoa were stored over time, ovarian complement proteins were progressively repressed by tryptophan and hippurate, indicating a remarkable adaptation of spermatozoa to the ovarian microenvironment. Before fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promotes spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism that influences the dynamics of spermatozoa in the female reproductive tract, providing valuable insights into the molecular networks regulating spermatozoa adaptation and survival in species with internal fertilization.

Effects of Fat and Carnitine on the Expression of Carnitine Acetyltransferase and Enoyl-CoA Hydratase Short-Chain 1 in the Liver of Juvenile GIFT (Oreochromis niloticus).

Carnitine acetyltransferase (CAT) and Enoyl-CoA hydratase short-chain 1 (ECHS1) are considered key enzymes that regulate the ß-oxidation of fatty acids. However, very few studies have investigated their full length and expression in genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China. Here, we cloned CAT and ECHS1 full-length cDNA via the rapid amplification of cDNA ends, and the expressions of CAT and ECHS1 in the liver of juvenile GIFT were detected in different fat and carnitine diets, as were the changes in the lipometabolic enzymes and serum biochemical indexes of juvenile GIFT in diets with different fat and carnitine levels. CAT cDNA possesses an open reading frame (ORF) of 2167 bp and encodes 461 amino acids, and the ECHS1 cDNA sequence is 1354 bp in full length, the ORF of which encodes a peptide of 391 amino acids. We found that juvenile GIFT had higher lipometabolic enzyme activity and lower blood CHOL, TG, HDL-C, and LDL-C contents when the dietary fat level was 2% or 6% and when the carnitine level was 500 mg/kg. We also found that the expression of ECHS1 and CAT genes in the liver of juvenile GIFT can be promoted by a 500 mg/kg carnitine level and 6% fat level feeding. These results suggested that CAT and ECHS1 may participate in regulating lipid metabolism, and when 2% or 6% fat and 500 mg/kg carnitine are added to the feed, it is the most beneficial to the liver and lipid metabolism of juvenile GIFT. Our results may provide a theoretical basis for GIFT feeding and treating fatty liver disease.

The Effect of Short-Term Artificial Feed Domestication on the Expression of Oxidative-Stress-Related Genes and Antioxidant Capacity in the Liver and Gill Tissues of Mandarin Fish (Siniperca chuatsi).

To investigate whether Mandarin fish developed oxidative stress after being domesticated with artificial feed, we conducted a series of experiments. Oxidative stress is an important factor leading to diseases and aging in the body. The liver integrates functions such as digestion, metabolism, detoxification, coagulation, and immune regulation, while the gills are important respiratory organs that are sensitive to changes in the water environment. Therefore, we used the liver and gills of Mandarin fish as research materials. The aim of this study was to investigate the effects of short-term artificial feed domestication on the expression of oxidative stress genes and the changes in oxidative-stress-related enzyme activity in the liver and gills of Mandarin fish. We divided the Mandarin fish into two groups for treatment. The control group was fed with live bait continuously for 14 days, while the experimental group was fed with half artificial feed and half live bait from 0 to 7 days (T-7 d), followed by solely artificial feed from 7 to 14 days (T-14 d). The experimental results showed that there was no difference in the body weight, length, and standard growth rate of the Mandarin fish between the two groups of treatments; after two treatments, there were differences in the expression of genes related to oxidative stress in the gills (keap1, kappa, gsta, gstt1, gstk1, SOD, and CAT) and in the liver (GPx, keap1, kappa, gsta, gstt1, gr, and SOD). In the liver, GPx activity and the content of MDA were significantly upregulated after 7 days of domestication, while in the gills, SOD activity was significantly upregulated after 7 days of domestication and GPx activity was significantly downregulated after 14 days of domestication. These results suggest that artificial feed domestication is associated with oxidative stress. Moreover, these results provide experimental basic data for increasing the production of aquaculture feed for Mandarin fish.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

Iopanoic acid alters thyroid hormone-related gene expression, thyroid hormone levels, swim bladder inflation, and swimming performance in Japanese medaka.

Disruption of the thyroid hormone system by synthetic chemicals is gaining attention owing to its potential negative effects on organisms. In this study, the effects of the dio-inhibitor iopanoic acid (IOP) on the levels of thyroid hormone and related gene expression, swim bladder inflation, and swimming performance were investigated in Japanese medaka. Iopanoic acid exposure suppressed thyroid-stimulating hormone ß (tshß), tshß-like, iodotyronin deiodinase 1 (dio1), and dio2 expression, and increased T4 and T3 levels. In addition, IOP exposure inhibited swim bladder inflation, reducing swimming performance. Although adverse outcome pathways of thyroid hormone disruption have been developed using zebrafish, no adverse outcome pathways have been developed using Japanese medaka. This study confirmed that IOP inhibits dio expression (a molecular initiating event), affects T3 and T4 levels (a key event), and reduces swim bladder inflation (a key event) and swimming performance (an adverse outcome) in Japanese medaka.

The genome regulatory landscape of Atlantic salmon liver through smoltification.

The anadromous Atlantic salmon undergo a preparatory physiological transformation before seawater entry, referred to as smoltification. Key molecular developmental processes involved in this life stage transition, such as remodeling of gill functions, are known to be synchronized and modulated by environmental cues like photoperiod. However, little is known about the photoperiod influence and genome regulatory processes driving other canonical aspects of smoltification such as the large-scale changes in lipid metabolism and energy homeostasis in the developing smolt liver. Here we generate transcriptome, DNA methylation, and chromatin accessibility data from salmon livers across smoltification under different photoperiod regimes. We find a systematic reduction of expression levels of genes with a metabolic function, such as lipid metabolism, and increased expression of energy related genes such as oxidative phosphorylation, during smolt development in freshwater. However, in contrast to similar studies of the gill, smolt liver gene expression prior to seawater transfer was not impacted by photoperiodic history. Integrated analyses of gene expression, chromatin accessibility, and transcription factor (TF) binding signatures highlight chromatin remodeling and TF dynamics underlying smolt gene regulatory changes. Differential peak accessibility patterns largely matched differential gene expression patterns during smoltification and we infer that ZNF682, KLFs, and NFY TFs are important in driving a liver metabolic shift from synthesis to break down of organic compounds in freshwater. Overall, chromatin accessibility and TFBS occupancy were highly correlated to changes in gene expression. On the other hand, we identified numerous differential methylation patterns across the genome, but associated genes were not functionally enriched or correlated to observed gene expression changes across smolt development. Taken together, this work highlights the relative importance of chromatin remodeling during smoltification and demonstrates that metabolic remodeling occurs as a preadaptation to life at sea that is not to a large extent driven by photoperiod history.

The azole biocide climbazole induces oxidative stress, inflammation, and apoptosis in fish gut.

Climbazole is an azole biocide that has been widely used in formulations of personal care products. Climbazole can cause developmental toxicity and endocrine disruption as well as gut disturbance in aquatic organisms. However, the mechanisms behind gut toxicity induced by climbazole still remain largely unclear in fish. Here, we evaluate the gut effects by exposing grass carp (Ctenopharyngodon idella) to climbazole at levels ranging from 0.2 to 20 µg/L for 42 days by evaluating gene transcription and expression, biochemical analyses, correlation network analysis, and molecular docking. Results showed that climbazole exposure increased cyp1a mRNA expression and ROS level in the three treatment groups. Climbazole also inhibited Nrf2 and Keap1 transcripts as well as proteins, and suppressed the transcript levels of their subordinate antioxidant molecules (cat, sod, and ho-1), increasing oxidative stress. Additionally, climbazole enhanced NF-κB and iκBα transcripts and proteins, and the transcripts of NF-κB downstream pro-inflammatory factors (tnfα, and il-1ß/6/8), leading to inflammation. Climbazole increased pro-apoptosis-related genes (fadd, bad1, and caspase3), and decreased anti-apoptosis-associated genes (bcl2, and bcl-xl), suggesting a direct reaction to apoptosis. The molecular docking data showed that climbazole could form stable hydrogen bonds with CYP1A. Mechanistically, our findings suggested that climbazole can induce inflammation and oxidative stress through CYP450s/ROS/Nrf2/NF-κB pathways, resulting in cell apoptosis in the gut of grass carp.

Uncoordinated 51-like kinase 1a/b and 2 in fish Megalobrama amblycephala: Molecular cloning, functional characterization, and their potential roles in glucose metabolism.

Uncoordinated (Unc) 51-like kinase (ulk1) and ulk2 are closely involved in autophagy activation, but little is known about their roles in regulating glucose homeostasis. In this study, the genes of ulk1a, ulk1b and ulk2 were cloned and characterized in fish Megalobrama amblycephala. All the three genes shared the approximate N-terminal kinase domain and the C-terminal Atg1-like_tMIT domain structure, while the amino acid sequence identity of them are different between M. amblycephala and other vertebrates. Their transcripts were widely observed in various tissues (brain, muscle, gill, heart, spleen, eye, liver, intestine, abdominal adipose and kidney), but differed in tissue expression patterns. During the glucose tolerance test and the insulin tolerance test, the up-regulated transcriptions of ulk1a, ulk1b and ulk2 were all found despite some differences in the temporal patterns. At the same time, the activities of glycolytic enzymes like hexokinase and phosphofructokinase both showed parallel increases. Furthermore, the feeding of a high-carbohydrate diet decreased the transcriptions of ulk1a, ulk1b and ulk2. Collectively, this study demonstrated that ulk1a, ulk1b and ulk2 in M. amblycephala had similar molecular characterizations, but with different conservation and tissue expression patterns. In addition, ulk1/2 might play important roles in maintaining the glucose homeostasis in fish through regulating the glycolytic pathway.

Spexin acts as a novel glucose-lowering factor in grass carp (Ctenopharyngodon idella).

At present, the physiological roles of various hormones in fish glucose metabolism have been elucidated. Spexin, a 14-amino acids polypeptide, is highly conserved in many species and has functions such as reducing body weight and improving insulin resistance. In this paper, the open reading frame (ORF) of spx21 in grass carp (Ctenopharyngodon idella) was cloned, and the tissue distribution of spx1 and spx2, their direct and indirect regulatory effects on glucose metabolism of grass carp were investigated. The ORF of spx2 gene in grass carp was 279 bp in length. Moreover, spx1 was highly expressed in the adipose tissue, while spx2 was highly expressed in the brain. In vitro, SPX1 and SPX2 showed opposite effects on the glycolytic pathway in the primary hepatocytes. In vivo, intraperitoneal injection of SPX1 and SPX2 significantly reduced serum glucose levels and increased hepatopancreas glycogen contents. Meanwhile, SPX1 and SPX2 promoted the expression of key genes of glycolysis (pk) and glycogen synthesis (gys) in the hepatopancreas at 3 h post injection. As for indirect effects, 1000 nM SPX1 and SPX2 significantly increased insulin-mediated liver type phosphofructokinase (pfkla) mRNA expression and enhanced the inhibitory effects of insulin on glucose-6-phosphatase (g6pase), phosphoenolpyruvate carboxykinase (pepck), glycogen phosphorylase L (pygl) mRNA expression. Our results show that SPX1 and SPX2 have similar indirect effects on the regulation of glucose metabolism that enhance insulin activity, but they exhibit opposite roles in terms of direct effects.

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.

Adverse effects of fenpropathrin on the intestine of common carp (Cyprinus carpio L.) and the mechanism involved.

Fenpropathrin (FEN), a pyrethroid pesticide, is frequently detected in natural water bodies, unavoidable pose adverse effects to aquatic organisms. However, the harmful effects and potential mechanisms of FEN on aquatic species are poorly understood. In this study, common carp were treatment with FEN at 0.45 and 1.35 µg/L for 14 d, and the toxic effects and underlying mechanisms of FEN on the intestine of carp were revealed. RNA-seq results showed that FEN exposure cause a wide range of transcriptional alterations in the intestine and the differentially expressed genes were mainly enrichment in the pathways related to immune and metabolism. Specifically, FEN exposure induced pathological damage and altered submicroscopic structure of the intestine, elevated the levels of Bacteroides fragilis enterotoxin, altered the contents of claudin-1, occludin, and zonula occluden-1 (ZO-1), and causing injury to the intestinal barrier. In addition, inflammation-related index TNF-α in the serum and IL-6 in the intestinal tissues were generally increased after FEN exposure. Moreover, FEN exposure promoted an increase in reactive oxygen species (ROS), altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), upregulated the contents of malondialdehyde (MDA) in the intestines. The apoptosis-related parameter cytochrome c, caspase-9, and caspase-3 were significantly altered, indicating that inflammation reaction, oxidative stress, and apoptosis may be involved in the toxic mechanism of FEN on carp. Moreover, FEN treatment also altered the intestinal flora community significantly, which may affect the intestinal normal physiological function and thus affect the growth of fish. Overall, the present study help to clarify the intestinal reaction mechanisms after FEN treatment, and provide a basis for the risk assessment of FEN.

USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1.

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.