Effects of cold acclimation on serum biochemical parameters and metabolite profiles in Schizothorax prenanti.

BACKGROUND: Environmental temperature is critical in regulating biological functions in fish. S. prenanti is a kind of cold-water fish, but of which we have little knowledge about the metabolic adaptation and physiological responses to long-term cold acclimation. RESULTS: In this study, we determined the physiological responses of S. prenanti serum after 30 days of exposure to 6℃. Compared with the control group, the levels of TC, TG, and LDL-C in the serum were significantly (P < 0.05) increased, and the level of glucose was significantly (P < 0.05) decreased under cold acclimation. Cold acclimation had no effect on the gene expression of pro-inflammatory factors and anti-inflammatory factors of S. prenanti. Metabolomics analysis by LC-MS showed that a total of 60 differential expressed metabolites were identified after cold acclimation, which involved in biosynthesis of amino acids, biosynthesis of unsaturated fatty acids, steroid degradation, purine metabolism, and citrate cycle pathways. CONCLUSION: The results indicate that cold acclimation can alter serum metabolites and metabolic pathways to alter energy metabolism and provide insights for the physiological regulation of cold-water fish in response to cold acclimation.

Unexpected regulatory functions of cyprinid Viperin on inflammation and metabolism.

BACKGROUND: Viperin, also known as radical S-adenosyl-methionine domain containing protein 2 (RSAD2), is an interferon-inducible protein that is involved in the innate immune response against a wide array of viruses. In mammals, Viperin exerts its antiviral function through enzymatic conversion of cytidine triphosphate (CTP) into its antiviral analog ddhCTP as well as through interactions with host proteins involved in innate immune signaling and in metabolic pathways exploited by viruses during their life cycle. However, how Viperin modulates the antiviral response in fish remains largely unknown. RESULTS: For this purpose, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the unique viperin gene has been knocked out by CRISPR/Cas9 genome-editing. In order to decipher the contribution of fish Viperin to the antiviral response and its regulatory role beyond the scope of the innate immune response, we performed a comparative RNA-seq analysis of viperin-/- and wildtype cell lines upon stimulation with recombinant fathead minnow type I interferon. CONCLUSIONS: Our results revealed that Viperin does not exert positive feedback on the canonical type I IFN but acts as a negative regulator of the inflammatory response by downregulating specific pro-inflammatory genes and upregulating repressors of the NF-κB pathway. It also appeared to play a role in regulating metabolic processes, including one carbon metabolism, bone formation, extracellular matrix organization and cell adhesion.

Intraspecific diversity loss in a predator species alters prey community structure and ecosystem functions.

Loss in intraspecific diversity can alter ecosystem functions, but the underlying mechanisms are still elusive, and intraspecific biodiversity-ecosystem function (iBEF) relationships have been restrained to primary producers. Here, we manipulated genetic and functional richness of a fish consumer (Phoxinus phoxinus) to test whether iBEF relationships exist in consumer species and whether they are more likely sustained by genetic or functional richness. We found that both genotypic and functional richness affected ecosystem functioning, either independently or interactively. Loss in genotypic richness reduced benthic invertebrate diversity consistently across functional richness treatments, whereas it reduced zooplankton diversity only when functional richness was high. Finally, losses in genotypic and functional richness altered functions (decomposition) through trophic cascades. We concluded that iBEF relationships lead to substantial top-down effects on entire food chains. The loss of genotypic richness impacted ecological properties as much as the loss of functional richness, probably because it sustains "cryptic" functional diversity.

Distribution and bioaccumulation of trace elements in two Cyprinidae fish species in the Indus river, Pakistan, including the impact of hydraulic structure on macroinvertebrates' biodiversity.

The concentration of trace elements (chromium, lead, zinc, copper, manganese, and iron) was determined in water, sediment and tissues of two Cyprinidae fish species - Labeo rohita and Tor putitora - collected from the eight sampling stations of Indus River in 2022 for four successive seasons (autumn, winter, spring, summer), and also study the present condition of macroinvertebrates after the construction of hydraulic structure. The obtained results of trace element concentrations in the Indus River were higher than the acceptable drinking water standards by WHO. The nitrate concentration ranges from 5.2 to 59.6 mg l-1, turbidity ranges from 3.00 to 63.9 NTU, total suspended solids and ammonium ions are below the detection limit (<0.05). In the liver, highest dry wt trace elements (µg/g) such as Cr (4.32), Pb (7.07), Zn (58.26), Cu (8.38), Mn (50.27), and Fe (83.9) for the Labeo rohita; and Tor Putitora has significantly greater accumulated concentration (Cr, Pb, Zn, Cu, Mn, Fe) in muscle and liver than did Labeo rohita species. Additionally, lower number of macroinvertebrates were recorded during the monsoonal season than pre-monsoon and post-monsoon. Local communities surrounded by polluted environments are more probably to consume more fish and expose them to higher concentrations of toxic trace elements (lead and copper). The findings also provide a basis for broader ecological management of the Indus River, which significantly influenced human beings and socioeconomic disasters, particularly in the local community.

Regulation of ddb2 expression in blind cavefish and zebrafish reveals plasticity in the control of sunlight-induced DNA damage repair.

We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.

The Response of the Gut Physiological Function and Microbiome of a Wild Freshwater Fish (Megalobrama terminalis) to Alterations in Reproductive Behavior.

The fish gut microbiome is well known for its role in degrading nutrients to improve the host's digestion and absorption efficiency. In this study, we focused on the core physiological adaptability during the various reproductive stages of the black Amur bream (Megalobrama terminalis) to explore the interaction mechanisms among the fish host gut mucosal structure, gut enzyme activity, and gut microbial metabolism in the course of the host's reproductive cycle. Our findings showed that M. terminalis exhibited locomotion metabolic type (aids in sporting) in the reproductive stage, and a change to visceral metabolic type (aids in digestion) during non-reproductive and post-reproductive stage phases. The impact of metabolic type selection and energy demand during various reproductive stages on fish nutrition strategy and digestive function was substantial. Our resulted showed that mitochondria in intestinal epithelial cells of reproductive M. terminalis appeared autophagy phenomenon, and the digestive enzyme activities in the intestines of reproductive M. terminalis were lower than those in the non-reproductive and post-reproductive individuals. Moreover, these differences in nutrition strategy have a prominent impact on the gut microbiome of reproductive M. terminalis, compared to non-reproductive and post-reproductive samples. Our findings showed that reproductive females had lower levels of alpha diversity compared to non-reproductive and post-reproductive females. Our results also showed a greater functional variety and an increase in functional genes related to carbohydrate, lipid, amino acid, cofactors, and vitamin metabolic pathways in the NRS and PRS group. It is noteworthy that an enrichment of genes encoding putative enzymes implicated in the metabolism of taurine and hypotaurine was observed in the RS samples. Our findings illustrated that the stability and resilience of the gut bacterial community could be shaped in the wild fish host-microbiome interactions during reproductive life history.

Interactive effects of temperature and protein levels on the growth performance, proximate composition, digestive enzyme activity and serum biochemistry of Labeo rohita.

The current study was performed to investigate the impact of different temperatures and protein levels on the growth performance, proximate composition and digestive and hepatic enzyme activities of Labeo rohita fingerlings. For this purpose, healthy fingerlings (average initial weight of 6.40 ± 0.02 g) were acclimatized for 15 days, then reared at three temperatures (25°C, 30°C and 35°C) and fed three levels of crude protein (25%, 30% and 35% crude protein (CP)) twice daily until satiation for 60 days. The results of the study revealed that the highest growth performance was observed in fish fed 35% protein and reared at 30°C. Similarly, fish reared at 35°C and 25°C water temperature showed comparatively better growth performance in fish fed with 35% protein. Furthermore, a significant enhancement in feed intake was observed with increasing culture temperature and increasing CP levels, but at 25°C, increasing CP levels significantly decreased the feed intake. Sligh variations were also observed in proximate composition in terms of moisture, CP, crude fat (CF) and ash contents in fish fed with different CP levels and reared at different temperatures. The hepatosomatic index and viscerosomatic index decreased significantly with increasing levels of protein and temperature. Amylase activities were significantly reduced with increasing culture temperature at each protein level. Increasing culture temperature did not affected the lipase activities. However, lipase activities were enhanced with increasing CP levels at 25°C and activities decreased with increasing CP levels at 30-35°C. Protease activity was enhanced with increasing temperature and CP levels. Significant increases were also observed in serum total proteins and liver functioning enzymes such as alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase in response to increased temperature, and protein had a reciprocal effect. It is concluded that increasing the CP levels increased the growth performance independent of temperature. However, similar growth performance at 30 CP (30°C) and 35 CP (35°C) indicates that L. rohita requires more protein at higher temperature for optimum growth.

Effects of a high-fat and high-carbohydrate diet on appetite regulation and central AMPK in the hypothalamus of blunt snout bream (Megalobrama amblycephala).

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor of cellular energy changes and controls food intake. This study investigates the effect of a high-calorie diet (high fat diet [HFD], high carbohydrate diet [HCD] and high energy diet [HED]) on appetite and central AMPK in blunt snout bream. In the present study, fish (average initial weight 45.84 ± 0.07 g) were fed the control, HFD, HCD and HED in four replicates for 12 weeks. At the end of the feeding trial, the result showed that body mass index, specific growth rate, feed efficiency ratio and feed intake were not affected (p > 0.05) by dietary treatment. However, fish fed the HFD obtained a significantly higher (p < 0.05) lipid productive value, lipid gain and lipid intake than those fed the control diet, but no significant difference was attributed to others. Also, a significantly higher (p < 0.05) energy intake content was found in fish-fed HFD, HCD and HED than those given the control diet. Long-term HFD and HCD feeding significantly increased (p < 0.05) plasma glucose, glycated serum protein, advanced glycation end product, insulin and leptin content levels than the control group. Moreover, a significantly lower (p < 0.05) complex 1, 2 and 3 content was found in fish-fed HFD and HCD than in the control, but no differences (p > 0.05) were attributed to those in HED. Fish-fed HED significantly upregulated (p < 0.05) hypothalamic ampα 1 and ampα 2 expression, whereas the opposite trend was observed in the hypothalamic mammalian target of rapamycin than those in HFD and HCD compared to the control. However, hypothalamic neuropeptide y, peroxisome proliferator-activated receptor α (pparα), acetyl-coa oxidase and carnitine palmitoyltransferase 1 were significantly upregulated (p < 0.05) in the HCD group, while the opposite was seen in cholecystokinin expression compared to those in the control group. Our findings indicated that the central AMPK signal pathway and appetite were modulated according to the diet's energy level to regulate nutritional status and maintain energy homoeostasis in fish.

Characterizing and identifying of miRNAs involved in berberine modulating glucose metabolism of Megalobrama amblycephala.

The present study, as one part of a larger project that aimed to investigate the effects of dietary berberine (BBR) on fish growth and glucose regulation, mainly focused on whether miRNAs involve in BBR's modulation of glucose metabolism in fish. Blunt snout bream Megalobrama amblycephala (average weight of 20.36 ± 1.44 g) were exposed to the control diet (NCD, 30% carbohydrate), the high-carbohydrate diet (HCD, 43% carbohydrate) and the berberine diet (HCB, HCD supplemented with 50 mg/kg BBR). After 10 weeks' feeding trial, intraperitoneal injection of glucose was conducted, and then, the plasma and liver were sampled at 0 h, 1 h, 2 h, 6 h, and 12 h. The results showed the plasma glucose levels in all groups rose sharply and peaked at 1 h after glucose injection. Unlike the NCD and HCB groups, the plasma glucose in the HCD group did not decrease after 1 h, while remained high level until at 2 h. The NCD group significantly increased liver glycogen content at times 0-2 h compared to the other two groups and then liver glycogen decreased sharply until at times 6-12 h. To investigate the role of BBR that may cause the changes in plasma glucose and liver glycogen, miRNA high-throughput sequencing was performed on three groups of liver tissues at 2 h time point. Eventually, 20 and 12 differentially expressed miRNAs (DEMs) were obtained in HCD vs NCD and HCB vs HCD, respectively. Through function analyzing, we found that HCD may affect liver metabolism under glucose loading through the NF-κB pathway; and miRNAs regulated by BBR mainly play roles in adipocyte lipolysis, niacin and nicotinamide metabolism, and amino acid transmembrane transport. In the functional exploration of newly discovered novel:Chr12_18892, we found its target gene, adenylate cyclase 3 (adcy3), was widely involved in lipid decomposition, amino acid metabolism, and other pathways. Furthermore, a targeting relationship of novel:Chr12_18892 and adcy3 was confirmed by double luciferase assay. Thus, BBR may promote novel:Chr12_18892 to regulate the expression of adcy3 and participate in glucose metabolism.

Recycling of animal protein wastes in the formulation of feed for Labeo rohita and Mystus vittatus-a comparative evaluation.

Lactic acid bacteria (LAB) are key players in the fermentation of organic wastes and their recycling as feedstuff for fish. Whey, a common dairy byproduct in India, is a cheap source of LAB and can be used to ferment animal byproducts. An experimental study was designed to explore whether the whey fermented animal protein blend (WFAPB) could be used as a fishmeal replacer in the formulation of feed for both stomach-less carp fish Labeo rohita and stomach-bearing catfish Mystus vittatus. Experiments were performed with five isoproteinous, isolipidous, and isoenergetic feeds with WFAPB replacing fishmeal (FM) by 0% (T1), 25% (T2), 50% (T3), 75% (T4), and 100% (T5). Fifteen days of laboratory experiments with these experimental feeds revealed that more than 50% FM replacement level could result in excess postprandial absorption (6 h) of some essential and non-essential amino acids in the plasma of both fish. The postprandial absorption was more in M. vittatus than L. rohita. Ninety-day experiments were conducted in outdoor cement vats to measure growths and deposition of amino acids (AA) in muscle. Regression analysis was performed to find the optimal FM replacement based on four growth parameters and fifteen AA deposition in muscle. A two-phase fuzzy methodology was used to obtain Pareto-optimal replacement levels for each fish. The results demonstrated that FM replacement levels were 7.63% and 36.79% respectively for L. rohita and M. vittatus when only four growth parameters were considered. However, based on the FM replacement level that maximized deposition of 15 amino acids and growth parameters, it was found that 12.23% and 40.02% replacement of FM by the WFAPB was ideal respectively for L. rohita and M. vittatus. The results revealed that only a fraction of both essential and non-essential amino acids absorbed in plasma could be converted into protein and deposited as bound amino acids in the muscle. It is concluded that fermentation by whey is an inexpensive, easily available, and environmentally sustainable technique to recycle animal protein in the formulation of feed for fish, and the stomach-bearing carnivorous fish are more efficient in utilizing fermented animal protein blend than the stomach-less carps.

Dietary berberine against intestinal oxidative stress, inflammation response, and microbiota disturbance caused by chronic copper exposure in freshwater grouper (Acrossocheilus fasciatus).

Berberine (BBR) is known for its strong antioxidant, anti-inflammatory, and capacity to preserve intestinal microbiota balance in fish. This study aimed to investigate the protective effects of berberine against copper-induced toxicity in the intestine of freshwater grouper Acrossocheilus fasciatus. The experiment involved four groups: a control group, a Cu group exposed to 0.02 mg/L Cu2+, and two BBR groups fed with 100 or 400 mg/kg of berberine diets and exposed to the same Cu2+ concentration. Three replicates of healthy fish (initial weight 1.56 ± 0.10 g) were subjected to their respective treatments for 30 days. Results showed that none of the treatments significantly affected the survival rate, final weight, weight gain, and feed intake (P > 0.05). However, supplementation with 100 and 400 mg/kg of BBR significantly lowered the antioxidant activities, and glutathione peroxidase (gpx) and superoxide dismutase (sod) expression levels, as well as reduced malondialdehyde (MDA) content caused by Cu2+ exposure (P < 0.05). Berberine inclusion significantly downregulated proinflammatory factors NLR family pyrin domain containing 3 (nlrp3), interleukin 1 beta (il1ß), interleukin 6 cytokine family signal transducer (il6st) but upregulated transforming growth factor beta 1 (tgfß1) and heat shock 70 kDa protein (hsp70) expression. Moreover, berberine at both levels maintained the intestinal structural integrity and significantly improved gap junction gamma-1 (gjc1) mRNA level compared to the Cu group (P < 0.05). Based on 16S rDNA sequencing, the richness and diversity of intestinal microbiota in different groups were not significantly influenced. Berberine reduced the Firmicutes/Bacteroidota ratio and stifled the growth of some specific pathogenic bacteria such as Pseudomonas, Citrobacter, and Acinetobacter, while boosting the richness of potential probiotic bacteria, including Roseomonas and Reyranella compared with the Cu group. In conclusion, berberine showed significant protective effects against Cu2+-induced intestinal oxidative stress, inflammation response, and microbiota disturbance in freshwater grouper.

Comprehensive transcripts analysis based on single-molecule real-time sequencing and Illumina sequencing provides insights into the mining of Toll-like receptor family in Schizothorax lissolabiatus.

Schizothorax lissolabiatus is an economically important cold-water fish species in southwestern China. Because of water pollution and habitat destruction, the number of wild populations has dramatically decreased. In this study, we used PacBio single-molecule real-time (SMRT) sequencing and Illumina sequencing to generate the first full-length transcriptome and transcriptome, respectively. A total of 19 310 polished consensus reads (PC) were obtained, with an average length of 1379 bp and an N50 length of 1485 bp. Meanwhile, 12 253 transcripts were successfully annotated as known homologous genes. The pathway annotation indicated that the enrichment and expression of most genes were mainly related to membrane, signal transduction and binding, and immune response. Furthermore, we identified 16 Toll-like receptors (TLRs) by mining the data from the transcripts. Phylogeny analysis showed that S. lissolabiatus TLR genes (slTLRs) supported the classification of TLRs into six families as in other vertebrates. Selection pressure analyses showed that 16 slTLRs revealed purification selection at the overall evolutionary selection. Further, positive selection signals were still detected in eight slTLRs, and most of the positive selection sites were located in the leucine-rich repeat region (LRR domain) associated with the recognition of pathogenic microorganisms, indicating that the function of these slTLR genes may be affected. Tissue specific expression analysis showed all slTLRs are present in kidney, spleen and liver but the relative expression varied among tissues. In conclusion, this study not only provided a valuable resource of transcripts for further research on S. lissolabiatus, but also contributed to improve the current understanding of the evolutionary history of immune-related genes and the TLR gene family in S. lissolabiatus.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially.

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 µL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 µL rOmhepc (100 µg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 µg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05). The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

Resveratrol attenuated oxidative stress and inflammatory and mitochondrial dysfunction induced by acute ammonia exposure in gibel carp (Carassius gibelio).

Ammonia is recognized as an environmental stressor for fish. As resveratrol (RES) has anti-inflammatory and antioxidant properties, we hypothesized that RES could attenuate the response to ammonia exposure in gibel carp. Therefore, gibel carp were fed a diet containing RES for eight weeks, followed by acute ammonia stimulation. Stress induced by acute ammonia exposure could be ameliorated by RES, manifested by down-regulated plasma glucose, and up-regulated C3 and IgM levels. Furthermore, decreased AST and LDH; enhanced T-AOC, SOD, and GPx in the liver; and reduced damage to gill and liver tissues indicated that RES attenuated oxidative and tissue damage induced by ammonia exposure. Moreover, RES activated the Nrf2/HO-1 pathway and up-regulated the expression of several antioxidant genes. RES enhanced anti-inflammatory activity as reflected by activation of the NF-κB pathway, down-regulated the expression of pro-inflammatory cytokines (nfκb, tnf-α, and il-1ß), and up-regulated the expression of anti-inflammatory cytokines (il-4 and il-10). In terms of mitochondrial function, RES up-regulated protein levels of p-AMPK, SIRT1, and PGC-1α; inhibited mitochondrial fission; promoted mitochondrial fusion and biogenesis-related gene expression. Overall, the results suggest that RES mediated the Nrf2/HO-1, NF-κB, and AMPK/SIRT1/PGC-1α pathways to attenuate oxidative stress, inflammation, and mitochondrial dysfunction induced by ammonia in gibel carp.

Redox Status, Immune Alterations, Histopathology, and Micronuclei Induction in Labeo rohita Dwelling in Polluted River Water.

In this study, we measured various parameters of oxidative stress, immune response, and abnormalities in the erythrocyte nucleus of Labeo rohita inhabiting the polluted Kshipra River, India. The river water contains heavy metals in this order: Ni > Fe > Cd > Cr > Mn > Zn > Cu. Fe showed the highest accumulation in gills, liver, and gut, whereas Ni (gills and gut) and Cd (liver) were lowest accumulated. The superoxide dismutase (SOD) and catalase (CAT) were found to be increased significantly (p < 0.05) in the gills (SOD: 211%; CAT: 150%), liver (SOD: 447%; CAT: 304%), and gut (SOD: 98.11%; CAT: 58.69%) in comparison with the reference fish. However, glutathione S transferase (GST) showed significantly (p < 0.05) higher activity in the gills (25.5%) but lower activity in the liver (- 49.22%) and the gut (- 30.57%). Moreover, reduced glutathione (GSH) decreased significantly (p < 0.05) in the gills (- 46.66%), liver (- 33.20%), and gut (- 39.87%). Despite the active response of the antioxidant enzymes, the highest lipid peroxidation was observed in the liver (463%). The effect of heavy metals was also observed on the immunity of the fish, causing immunosuppression as evident by significantly (p < 0.05) lower values of acid phosphatase (- 50%), myeloperoxidase (- 48.33%), and nitric oxide synthase (- 50%) in serum. Histopathological findings showed gill lamellae shortening, hyperplasia, club-shaped lamellar tip in exposed gills and necrosis, vacuolization, and pyknosis in the exposed liver. Furthermore, polluted river water was also found to induce micronuclei (2.1%) and lobed nuclei (0.72%) in erythrocytes (0.65%). These results indicate the potential of heavy metal-induced oxidative stress and other forms of stress in inhabiting fish, highlighting the need to control the pollution of this river water.

Cardiac Hypoxia Tolerance in Fish: From Functional Responses to Cell Signals.

Aquatic animals are increasingly challenged by O2 fluctuations as a result of global warming, as well as eutrophication processes. Teleost fish show important species-specific adaptability to O2 deprivation, moving from intolerance to a full tolerance of hypoxia and even anoxia. An example is provided by members of Cyprinidae which includes species that are amongst the most tolerant hypoxia/anoxia teleosts. Living at low water O2 requires the mandatory preservation of the cardiac function to support the metabolic and hemodynamic requirements of organ and tissues which sustain whole organism performance. A number of orchestrated events, from metabolism to behavior, converge to shape the heart response to the restricted availability of the gas, also limiting the potential damages for cells and tissues. In cyprinids, the heart is extraordinarily able to activate peculiar strategies of functional preservation. Accordingly, by using these teleosts as models of tolerance to low O2, we will synthesize and discuss literature data to describe the functional changes, and the major molecular events that allow the heart of these fish to sustain adaptability to O2 deprivation. By crossing the boundaries of basic research and environmental physiology, this information may be of interest also in a translational perspective, and in the context of conservative physiology, in which the output of the research is applicable to environmental management and decision making.

Comparative Expression Profiling Reveals the Regulatory Effects of Dietary Mannan Oligosaccharides on the Intestinal Immune Response of Juvenile Megalobrama amblycephala against Aeromonas hydrophila Infection.

Mannan oligosaccharides (MOS) are functional oligosaccharides with beneficial effects on the non-specific immunity of Megalobrama amblycephala, but systematic studies on the immunomodulatory mechanisms of MOS are still lacking. To investigate the protective mechanisms of three different levels of dietary MOS supplementation on the intestinal immunity of juvenile M. amblycephala, comparative digital gene expression (DGE) profiling was performed. In this study, 622 differentially expressed genes (DEGs) were identified, while the similar expression tendency of 34 genes by qRT-PCR validated the accuracy of the DGE analyses. Gene Ontology (GO) enrichment revealed that the DEGs were mainly enriched in two functional categories of biological process and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly related to complement and coagulation cascades, coagulation cascades, platelet activation, natural killer cell mediated cytotoxicity, Fc gamma R-mediated phagocytosis and antigen processing and presentation. In addition, the pro-inflammatory, apoptosis and tight junction-related genes were more significantly up-regulated upon infection in the dietary MOS groups to enhance host immune functions and maintain the stability of the intestinal barrier. These results will be helpful to clarify the regulatory mechanism of MOS on the intestinal immunity of M. amblycephala and lay the theoretical foundation for the prevention and protection of fish bacterial diseases.

Vascular Endothelial Growth Factor A (VEGFA) Regulates Hepatic Lipid and Glycogen Metabolism in Schizothorax prenanti.

Vascular endothelial growth factor A (VEGFA) plays important roles in angiogenesis, inflammatory response as well as energy metabolism in mammals. However, its effect on glycolipid metabolism in fish has not been reported. In this study, we cloned and characterized the vegfa gene of Schizothorax prenanti (S. prenanti). vegfa expression was significantly higher in liver and muscle than that in other tissues. Then, the VEGFA recombinant protein was expressed in Escherichia coli and obtained after purification. VEGFA i.p. injection significantly increased the serum glucose and TG content compared with the control group. Moreover, VEGFA protein aggravated the glycogen and lipid deposition in the liver of S. prenanti. In addition, we found that VEGFA treatment increased hepatocyte glycogen and lipid droplet content and increased the levels of pAMPKα (T172). Furthermore, AMPKα inhibition attenuated the ability of VEGFA to induce TG and glycogen accumulation. These results demonstrate that VEGFA regulates hepatic lipid and glycogen metabolism through AMPKα in S. prenanti, which may contribute to a better understanding of VEGFA functions in the glycolipid metabolism of fish.

Protective effects of the fructooligosaccharide on the growth performance, biochemical indexes, and intestinal morphology of blunt snout bream (Megalobrama amblycephala) infected by Aeromonas hydrophila.

The purpose of the study was to investigate the effects of dietary fructooligosaccharide (FOS) on growth performance, biochemical indexes, intestinal morphology, and growth-related gene expression of blunt snout bream (Megalobrama amblycephala) infected by Aeromonas hydrophila (AH). Two hundred twenty-five healthy blunt snout bream with an initial body weight of 38.41 ± 0.88 g were randomly divided into five groups with three replicates: control (basal diet), model (AH + basal diet), SFOS (AH + 2 g/kg FOS), MFOS (AH + 4 g/kg FOS), LFOS (AH + 6 g/kg FOS). After 9 weeks of feeding, the results showed that the FOS-added diet abrogated AH-induced retardation, hemorrhage, and inflammatory infiltration. FOS supplementation enhanced the growth performance degradation caused by AH, and the highest growth performance was observed at MFOS. Meanwhile, the addition of FOS to feed improved the blood immunity reduced by AH. In expansion, the mucosal epithelium of intestinal villi exfoliated, exposing the lamina propria, and a few villi were genuinely harmed in the model group. Fish fed with MFOS ameliorated the damaged intestine, evidenced by well-preserved intestine architecture. Furthermore, the model group downregulated the expression of growth-related genes (growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-1)). Fish fed with 2 g/kg or 4 g/kg FOS upregulated the genes specified above expressions in the liver compared with the model group. In conclusion, the results mentioned above suggested that the dietary FOS could relieve the pressure to elevate the immune damage and intestine injury induced by AH and enhance the hepatic expression of IGF-1 and GHR.

Functional exploration of SNP mutations in HIF2αb gene correlated with hypoxia tolerance in blunt snout bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A203A752) and its homozygous diplotype II (A203A203A752A752) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOEcrit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48 h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48 h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.