Production of Antioxidant, Angiotensin-Converting Enzyme Inhibitory and Osteogenic Gelatin Hydrolysate from Labeo rohita Swim Bladder.

Optimization of antioxidants and angiotensin-converting enzyme (ACE) inhibitory potential gelatin hydrolysate production from Labeo rohita (rohu) swim bladder (SBGH) by alcalase using central composite design (CCD) of response surface methodology (RSM) was investigated. The maximum degree of hydrolysis (DH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), total antioxidants (TAO), and ACE inhibitory activity were achieved at 0.1:1.0 (w/w) enzyme to substrate ratio, 61 °C hydrolysis temperature, and 94-min hydrolysis time. The resulting SBGH obtained at 19.92% DH exhibited the DPPH (24.28 µM TE/mg protein), ABTS (34.47 µM TE/mg protein), TAO (12.01 µg AAE/mg protein), and ACE inhibitory (4.91 µg/mg protein) activity. Furthermore, SBGH at 100 µg/ml displayed osteogenic property without any toxic effects on MC3T3-E1 cells. Besides, the protein content of rohu swim bladder gelatin (SBG) and SBGH was 93.68% and 94.98%, respectively. Both SBG and SBGH were rich in glycine, proline, glutamic acid, alanine, arginine, and hydroxyproline amino acids. Therefore, SBGH could be an effective nutraceutical in functional food development.

Dynamic alterations in physiological and biochemical indicators of Cirrhinus mrigala hatchlings: A sublethal exposure of triclosan.

Triclosan (TCS), a biocide used in various day-to-day products, has been associated with several toxic effects in aquatic organisms. In the present study, biochemical and hematological alterations were evaluated after 14 d (sublethal) exposure of tap water (control), acetone (solvent control), 5, 10, 20, and 50 µg/L (environmentally relevant concentrations) TCS to the embryos/hatchlings of Cirrhinus mrigala, a major freshwater carp distributed in tropic and sub-tropical areas of Asia. A concentration-dependent increase in the content of urea and protein carbonyl, while a decrease in the total protein, glucose, cholesterol, triglycerides, uric acid, and bilirubin was observed after the exposure. Hematological analysis revealed a decrease in the total erythrocyte count, hemoglobin, and partial pressure of oxygen, while there was an increase in the total leucocyte count, carbon dioxide, and partial pressure of carbon dioxide and serum electrolytes. Comet assay demonstrates a concentration-dependent increase in tail length, tail moment, olive tail moment, and percent tail DNA. An amino acid analyzer showed a TCS-dose-dependent increase in various amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed different proteins ranging from 6.5 to 200 kDa, demonstrating TCS-induced upregulation. Fourier transform infrared spectra analysis exhibited a decline in peak area percents with an increase in the concentration of TCS in water. Curve fitting of amide I (1,700-1600 cm-1) showed a decline in α-helix and turns and an increase in ß-sheets. Nuclear magnetic resonance study also revealed concentration-dependent alterations in the metabolites after 14 d exposure. TCS caused alterations in the biomolecules and heamatological parameters of fish, raising the possibility that small amounts of TCS may change the species richness in natural aquatic habitats. In addition, consuming TCS-contaminated fish may have detrimental effects on human health. Consequently, there is a need for the proper utilisation and disposal of this hazardous compound in legitimate quantities.

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.

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.

Proteome changes in larval zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) exposed to (±) anatoxin-a.

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following  (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01-3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

Regulation of sirt1 and foxO1 in glucose metabolism of Megalobrama amblycephala.

Both silent information regulator 2 homolog 1 (sirt1) and forkhead box transcription factor 1 (foxO1) are crucial transcription factors involved in glucolipid metabolism and energy regulation. The presnt study aimed to understand their regulatory roles in glucose metabolism. Molecular cloning and sequencing of sirt1 gene of Megalobrama amblycephala (masirt1) was conducted and cellular localization of both the factors were analysed. Their effects and action patterns in the glucose metabolism of Megalobrama amblycephala (M. amblycephala) were investigated through acute and long-term glucose tolerance assays. The results revealed that the full-length masirt1 cDNA sequence was 2350 bp and closely related to Sinocyclocheilus rhinocerous. Sirt1 and foxO1 were found to be mutually dependent and localized in the nucleus. Acute glucose tolerance tests revealed that the expression levels of both factors in the liver of M. amblycephala showed an initial increase followed by a decrease. Plasma glucose levels in M. amblycephala significantly increased at 2 and 12 h (P < 0.05). In a long-term breeding experiment with high-sugar feeding, the expressions of the sirt1 and foxO1 genes in the kidney and intestine of M. amblycephala exhibited synergistic changes. The 51WS groups had significantly higher levels of sirt1 and foxO1 gene expression in the kidney and intestine compared to the 0WS and 17WS groups (P < 0.05). Overall, masirt1 is evolutionarily highly conserved, and the interaction site of sirt1 and foxO1 is located in the nucleus. In long-term hyperglycemic regulation, sirt1 and foxO1 exhibit synergistic regulatory effects in the kidney and intestine of M. amblycephala. This study provides insights into how sirt1 and foxO1 regulate glucose metabolism in M. amblycephala.

Alteration of haematological and biochemical biomarkers after sub-lethal chronic malathion (Elathion®) intoxication in freshwater fish, Labeo rohita (Hamilton, 1822).

The present investigation aimed to evaluate the long-term effects of malathion (Elathion®) at two sub-lethal concentrations (0.36 and 1.84 mgL-1) for 45 days after the determination of 96 h-LC50 value (18.35 mgL-1) in a commercially important aquaculture species, Labeo rohita by assaying multiple biomarker approaches. Total erythrocyte count (TEC), and haemoglobulin count (Hb) were found to be decreased while total leucocyte counts (TLC) were increased (p < 0.05) in malathion-intoxicated fish. Malathion exposure significantly reduced (p < 0.05) serum protein levels while significantly increased (p < 0.05) blood glucose levels. RNA activity in muscle was reduced (p < 0.05) while DNA activity increased (p < 0.05) in malathion-intoxicated fish. Acid phosphatase (ACP) activities in the brain; lacate dehydrogenase (LDH) activities in brain and liver were increased (p < 0.05), while alkaline phosphatase (ALP) activities in the brain; succinate dehydrogenase (SDH) activities in the brain, liver and kidney; acetylcholine esterase (AChE) activity in the brain; and ATPase activities in the brain, liver and kidney were reduced (p < 0.05) in comparison to control. Thus, the alteration in studied biomarkers was in a concentation-time dependent manner; however, it was more pronounced at the higher concentration at 45 days of exposure. The alteration in biomarker activity is probably a defensive mechanism/ adaptive response of fish to overcome the stress induced by malathion, which is a novel insight and possible impact on L.rohita. Our findings suggest malathion-induced stress, therefore, the use of malathion needs to be regulated to safeguard aquatic animals including fish and human health.

Therapeutic Role of Hemp (Cannabis sativa) Against Copper-Induced Toxicity in Labeo rohita and Cirrhinus mrigala.

For decades hemp has been used as a therapeutic agent for enhancing immunity in animals. Current study was conceptualized to find out the protective role of dietary hemp seed products (hemp seed oil (HO) and hemp seed (HS)) against copper-induced toxicity in fish. Fingerlings of Labeo rohita (Rohu) and Cirrhinus mrigala (Mrigal) were exposed to copper at 20% of the 96 h LC50 (1.34 and 1.52 ppm, respectively) for 30 days. Following Cu exposure, fish were maintained on two types of hemp (Cannabis sativa)-supplemented feeds, on graded levels of hemp seed oil (HO: 1%, 2%, 3%) and hemp seed (HS: 5%, 10%, 15%) for 50 days, while one group was the control (without any copper exposure as well as any supplementation). Copper exposure significantly increased (P < 0.05) WBCs, hematocrit, MCHV, eosinophils, and lymphocytes in L. rohita and also in C. mrigala as compared to control. Copper exposure also significantly (P < 0.05) changed lysozymes, plasma protein, and IgM in both species, in comparison to control. Moreover, alkaline phosphatase, bilirubin, serum glutamic-pyruvic transaminase, and aspartate transaminase were significantly (P < 0.05) changed by copper exposure in comparison to control in both species. Additionally, Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase were also significantly (P < 0.05) increased in the brain, gills, liver, and muscle of copper-exposed group in both species as compared to control. Interestingly, all the altered parameter of blood, serum, liver function tests, and antioxidant enzymes (in different organs) because of copper toxicity were successfully reverted to normal level in hemp seed oil (HO) and hemp seed (HS)-supplemented fed groups of both species. In conclusion, hemp seed supplementation showed significant (P < 0.05) improved results against copper toxicity. Thus, it could be recommended as an animal feed ingredient for its therapeutic role.

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.

Antioxidant enzyme activities, molecular docking studies, MM-GBSA, and molecular dynamic of chlorpyrifos in freshwater fish Capoeta umbla.

Chlorpyrifos (CPF), which was started to be used in 1965, is a broad spectrum organophosphate insecticide that is used more and more day by day. Commonly used to control pests in farmland and homes, CPF is more toxic to fish than organochlorine compounds. CPF poses a serious threat to the health of humans and aquatic organisms. This paper studies the relationship between CPF exposure and antioxidant enzyme activities in gill, kidney and liver tissues of Capoeta umbla. Different time intervals (12, 24, 48, 72, and 96 h) and CPF doses (55 and 110 µg L-1) were used in the study. Spectrophotometrical measures were taken in all tissues for antioxidant enzyme activities and malondialdehyde (MDA) levels, as indices of the lipid peroxidation (LPO). A positive relationship between CPF and MDA levels was found in the study at a statistically significant level (p < 0.05). The study also found a negative relationship between CPF levels and catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activity. Independent variables in the study can act as biomarkers of CPF exposure. The study recommends employing proper ecotoxicological risk evaluations in cases of CPF usage as a pesticide. The activities of the studied molecules against various proteins that are crystal structure of human peroxiredoxin 5 (PDB ID: 1HD2) has docking score value is -2.67, crystal structure of Bovine Xanthine Oxidase (PDB ID: 3NRZ) has docking score value is -3.76, and crystal structure of antibacterial FabH (PDB ID: 4Z8D) has docking score value is -3.16, were compared. Molecular dynamic (MD) calculations were made in 100 ns. MM/GBSA methods are calculated binding free energy. Afterwards, ADME/T analysis was performed to examine the some properties of the molecules.Communicated by Ramaswamy H. Sarma.

Hazard assessment of complex legacy-contaminated groundwater mixtures using a novel approach method in adult fathead minnows.

Traditional risk assessment methods face challenges in the determination of drivers of toxicity for complex mixtures such as those present at legacy-contaminated sites. Bioassay-driven analysis across several levels of biological organization represents an approach to address these obstacles. This study aimed to apply a novel transcriptomics tool, the EcoToxChip, to characterize the effects of complex mixtures of contaminants in adult fathead minnows (FHMs) and to compare molecular response patterns to higher-level biological responses. Adult FHMs were exposed for 4 and 21 days to groundwater mixtures collected from a legacy-contaminated site. Adult FHM showed significant induction of micronuclei in erythrocytes, decrease in reproductive capacities, and some abnormal appearance of liver histology. Parallel EcoToxChip analyses showed a high proportion of upregulated genes and a few downregulated genes characteristic of compensatory responses. The three most enriched pathways included thyroid endocrine processes, transcription and translation cellular processes, and xenobiotics and reactive oxygen species metabolism. Several of the most differentially regulated genes involved in these biological pathways could be linked to the apical outcomes observed in FHMs. We concluded that molecular responses as determined by EcoToxChip analysis show promise for informing of apical outcomes and could support risk assessments of complex contaminated sites.

An experiment on the glucose metabolite, serum electrolytes, and somatic characteristics of the Levantine Barbel Luciobarbus pectoralis (Heckel, 1843) under the effect of heavy metals.

Levantine Barbel (Luciobarbus pectoralis) is a benthopelagic, subtropical native fish living in the inland waters of the Mediterranean region in Türkiye and Syria. Even though it is widely consumed locally, experimental observations on how heavy metals [zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb)] and their mixtures affect the fish are lacking. Several bioindicators of the fish exposed to heavy metals are the focus of the current investigation. Initially, Fulton condition factor (K) and hepato-somatic index (HSI) were utilized in the somatic characteristics of L. pectoralis. Then, changes in the level of glucose metabolite and electrolytes [sodium (Na+), potassium (K+), and chloride (Cl-)] of blood were determined by Architect C-800 auto-analyzer after exposure durations. The results of the experiments demonstrated that heavy metals can rapidly have a negative impact on the regulation of blood and somatic characteristics of fish. It was observed that the K index decreased in all metal groups at 24 and 96 h, while considerably increased in the 24-h effect of cadmium only (P ≤ 0.05). Along with that, in the 96-h effect of metals, Cu indicated the highest decrease in the HSI value (19.33%, P ≤ 0.05). In general, all heavy metal exposures caused the fish's glucose metabolite level to rise compared to the control (P ≤ 0.05). Furthermore, sublethal effects of metals at both durations caused considerable changes in blood electrolytes of the fish compared to control (P ≤ 0.05). Additionally, putative biomarkers in both durations had the greatest difference in toxic similarity under the Cu impact compared to the control, according to Hierarchical clustering and Euclidean distance metrics. Although the applied concentrations of Zn, Cu, Cd, and Pb and their mixture studied were generally within the limits of the various organizations and the surface water regulations, changes in ecophysiological and somatic indices were nonetheless seen in fish.

Characterization of molecular and apical effects of legacy-contaminated groundwater on early life stages of fathead minnows.

Mechanistic toxicology approaches represent a promising alternative to traditional live animal testing; however, the often-noted uncertainties concerning the linkages between effects observed at molecular and apical levels curtails the adoption of such approaches. The objective of this study was to apply a novel transcriptomics tool, EcoToxChips, to characterize the effects of complex mixtures of contaminants in fish and to compare molecular response patterns to higher-level biological responses including swimming behavior, deformities, and mortality. Fathead minnow (FHM) embryos were exposed for seven days to increasing concentrations of groundwater collected from moderate (MIAZ) and high (HIAZ) industrial activity zones of a legacy contaminated site. There was a concentration-dependent disruption of photo-dependent swimming responses associated with avoidance behavior patterns and spinal deformities (HIAZ and MIAZ), and an induction of pericardial edema and mortality (HIAZ-10%). Parallel EcoToxChip analyses showed a shift from a majority of upregulated genes at lower concentrations to a majority of downregulated genes at higher concentrations for both treatment conditions. Many of the significantly differentially regulated genes were involved in biological pathways including induction of oxidative stress, activating of several metabolic processes and growth, cell death, and inhibition of signal transduction signaling processes. Several contaminants present in the groundwater mixtures could have contributed to an exceedance of antioxidant system capacities that possibly led to the deformities, altered swimming behaviours, and mortality observed in FHMs. Therefore, molecular response patterns could be linked to apical outcomes observed in this study. Overall, the results observed in this study demonstrate that transcriptomics approaches such as the EcoToxChip system could be supportive of risk assessment of complex contaminated sites.

Potentials of Aloe barbadensis inclusion in fish feeds on resilience to Aeromonas hydrophila infection in freshwater fish Labeo rohita.

Aquatic bacterial pathogens can cause severe economic loss in aquaculture industry. An opportunistic pathogen, Aeromonas hydrophila is responsible for Motile Aeromonas Septicemia, leading to high mortality rates in fish. The present study was focused on the efficacy of Aloe barbadensis replacing fishmeal diets on hematological, serum biochemical, antioxidant, histopathological parameters, and disease resistance against A. hydrophila infection in Labeo rohita. Isonitrogenous fishmeal replaced diets (FMR) were prepared with varying levels of A. barbadensis at D1 (0%) (control), D2 (25%), D3 (50%), D4 (75%) and D5 (100%) then fed to L. rohita. After 60 days of post-feeding, the experimental fish were challenged with A. hydrophila. Blood and organs were collected and examined at 1- and 15-days post infection (dpi). The results demonstrated that on 1 dpi, white blood cells (WBC), total protein, cholesterol and low-density lipoprotein (LDL) levels were significantly increased in D3 diet fed groups. The D2 and D3 diet fed group showed decreasing trends of serum glutamic pyruvic transaminase (SGPT) and antioxidant enzymes activity on 15 dpi. The histopathological architecture results clearly illustrated that the D3 diet fed group had given a higher protective effect by reducing the pathological changes associated with A. hydrophila infection in liver, intestine and muscle. Higher percentage of survival rate was also observed in D3 diet fed group. Therefore, the present study suggested that the dietary administration of A. barbadensis up to 50% fishmeal replacement (D3 diet) can elicit earlier antioxidant activity, innate immune response and improve survival rate in L. rohita against A. hydrophila infection.

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.

Effect of glyphosate-based herbicide roundup on hemato-biochemistry of Labeo rohita (Hamilton, 1822) and susceptibility to Aeromonas hydrophila infection.

In the present study, comprehensive research was executed to investigate the salient toxic effects of glyphosate herbicide in static water system by evaluating the haemato-biochemical profiles of Labio rohita. A challenge study against Aeromonas hydrophila was conducted to determine disease susceptibility of the fish, treated to varying concentrations of commercial-grade glyphosate herbicide. A static range finding bioassay and definitive test revealed that the 96-h LC50 value of glyphosate was 10.16 mg L-1. The experimental fish were subjected to three sub-lethal concentrations of 2.06, 1.03, and 0.63 mg l-1 for 28 days and changes were documented bi-fortnightly to study haemato-biochemical alterationsin the fish. Significantly (p < 0.05) low values in red blood corpuscles (RBC), hemoglobin (Hb), and hematocrit value (Hct) were documented. In contrast, a significant (p < 0.05) escalation in white blood corpuscles (WBC) was documented in comparison to the control. Biochemical and stress markers such as blood glucose, total protein, and alkaline phosphatase (ALP) were significantly (p < 0.05) low, whereas serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) escalated significantly (p < 0.05). Chronic exposure to glyphosate, on the other hand, had the least effect on the Na+ and K+ ions. Further, a challenge assay against A. hydrophila at three sub-lethal glyphosate concentrations demonstrated a synergistic impact that reduced the fish survivability. The findings conclude that persistent low glyphosate concentrations in aquatic ecosystems show significant pathophysiological changes in L. rohita, with increased vulnerability to infections. Altogether, our findings indicate the need to further study the possible assessment for a sustainable bio-remediation technique, mitigation of the detrimental effects of glyphosate exposure in fish, and recommendation of an acceptable residue concentration of the glyphosate in aquatic ecosystem.

High-fat diet alters intestinal microbiota and induces endoplasmic reticulum stress via the activation of apoptosis and inflammation in blunt snout bream.

The primary organ for absorbing dietary fat is the gut. High dietary lipid intake negatively affects health and absorption by causing fat deposition in the intestine. This research explores the effect of a high-fat diet (HFD) on intestinal microbiota and its connections with endoplasmic reticulum stress and inflammation. 60 fish (average weight: 45.84 ± 0.07 g) were randomly fed a control diet (6% fat) and a high-fat diet (12 % fat) in four replicates for 12 weeks. From the result, hepatosomatic index (HSI), Visceralsomatic index (VSI), abdominal fat (ADF), Intestosomatic index (ISI), mesenteric fat (MFI), Triglycerides (TG), total cholesterol (TC), non-esterified fatty acid (NEFA) content were substantially greater on HFD compared to the control diet. Moreover, fish provided the HFD significantly obtained lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities. In contrast, an opposite result was seen in malondialdehyde (MDA) content in comparison to the control. HFD significantly altered intestinal microbiota in blunt snout bream, characterized by an increased abundance of Aeromonas, Plesiomonas proteobacteria, and firmicutes with a reduced abundance of Cetobacterium and ZOR0006. The transcriptional levels of glucose-regulated protein 78 (grp78), inositol requiring enzyme 1 (ire1), spliced X box-binding protein 1 (xbp1), DnaJ heat shock protein family (Hsp40) member B9 (dnajb9), tumor necrosis factor alpha (tnf-α), nuclear factor-kappa B (nf-κb), monocyte chemoattractant protein-1 (mcp-1), and interleukin-6 (il-6) in the intestine were markedly upregulated in fish fed HFD than the control group. Also, the outcome was similar in bax, caspases-3, and caspases-9, ZO-1, Occludin-1, and Occludin-2 expressions. In conclusion, HFD could alter microbiota and facilitate chronic inflammatory signals via activating endoplasmic reticulum stress.

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.

Effects of hypoxia on the gill morphological structure, apoptosis and hypoxia-related gene expression in blunt snout bream (Megalobrama amblycephala).

The blunt snout bream (Megalobrama amblycephala) is a typical hypoxia-sensitive fish, and hypoxia stress leads to reduced vitality and yield during aquaculture. To explore the specific adaptation mechanism under hypoxia, the blunt snout bream was treated with hypoxia (DO = 2.0 ± 0.1 mg/L) for 24 h, followed by 3 h of recovery. Our results depicted that the gill filament structure of blunt snout bream changed after hypoxia. During hypoxia for 24 h, the gill filament structure was altered, including a more than 80% expansion of the lamellar respiratory surface area and a proportionate apoptosis decrease in interlamellar cell mass (ILCM) volume. Thus, the water-blood diffusion distance was shortened to less than 46%. During hypoxia for 24 h, the activity of ROS in gill tissue increased significantly (p < 0.05), while the mitochondrial membrane potential decreased significantly (p < 0.05). During hypoxia, mRNA expression level of anti-apoptotic gene Bcl-2 in the gills of blunt snout bream decreased significantly (p < 0.05), while the expression of pro-apoptotic gene Bax mRNA increased significantly (p < 0.05). Thus, the ratio of Bax/Bcl-2 mRNA increased in the gills of blunt snout bream to promote the activity of Caspase-3. Together, our results indicated hypoxia-induced apoptosis in the gills of blunt snout bream through the mitochondrial pathway. In addition, a decreased expression of Phd1 and an increased expression of Hif-1α in gills under hypoxia stress indicates that blunt snout bream may cope with hypoxia-induced apoptosis by enhancing the HIF pathway. These results provide new insights into fish's adaptation strategies and mechanisms of hypoxia.