Hydrophila Challenge Research Articles

Molecular cloning, tissues distribution, and function analysis of thioredoxin-like protein-1 (TXNL1) in Chinese giant salamanders Andrias davidianus

Thioredoxin-like protein-1 (TXNL1) is the member of thioredoxin superfamily, a family of thiol oxidoreductases. TXNL1 plays an important role in scavenging ROS and the maintenance of cellular redox balance. However, its physiological functions in Andrias davidianus have not been well understood. In the present study, the full-length cDNA encoding thioredoxin-like protein-1 (AdTXNL1) of A. davidianus was cloned, the mRNA tissue distribution was analyzed, and the function was characterized. The Adtxnl1 cDNA contained an open reading frame (ORF) of 870 bp encoding a polypeptide of 289 amino acids with the N-terminal TRX domain, a Cys34-Ala35-Pro36-Cys37 (CAPC) motif, and the C-terminal proteasome-interacting thioredoxin domain (PITH). The mRNA of AdTXNL1 was expressed in a wide range of tissues, with the highest level in the liver. The transcript level of AdTXNL1 was significantly up-regulated post Aeromonas hydrophila challenge in liver tissue. Moreover, the recombinant AdTXNL1 protein was produced and purified, and used to investigate the antioxidant activity. In the insulin disulfide reduction assay, rAdTXNL1 exhibited strong antioxidant capability. Altogether, the thioredoxin-like protein-1 may be involved in reduction/oxidation (redox) balance and as an important immunological gene in A. davidianus.

Effects of Iron and Vitamin C on Growth Performance, Iron Utilization, Antioxidant Capacity, Nonspecific Immunity, and Disease Resistance to Aeromonas hydrophila in Chinese Mitten Crab (Eriocheir sinensis)

Iron is an essential trace element with an abroad physiological function, and iron deficiency can impair animal health. Vitamin C (VC) has the potential to release iron from diets and increase iron uptake. This study evaluates the effects of dietary iron and vitamin C on the growth performance, iron utilization, antioxidant capacity, and nonspecific immunity of the Chinese mitten crab (Eriocheir sinensis). Juvenile E. sinensis ( 1.14 ± 0.01 g ) were fed six diets supplemented with three levels of iron (41.40, 92.25, and 143.00 mg/kg), and each iron level supplemented either 4.33 mg/kg or 700.90 mg/kg of vitamin C for 8 weeks. Crabs fed 41.40 mg/kg of iron diet had the lowest weight gain (WG) and specific growth rate (SGR) and had the highest feed conversion rate (FCR) regardless of dietary vitamin C levels. Dietary 700.90 mg/kg of vitamin C improved WG and SGR but decreased FCR when the diets were supplemented with 41.40 and 143.00 mg/kg of iron ( P < 0.05 ). Dietary 700.90 mg/kg of vitamin C significantly improved the expression levels of divalent metal ion transporter 1, ferroportin, and iron regulation protein in the intestine and hepatopancreas when supplemented with 92.25 and 143.00 mg/kg of iron ( P < 0.05 ) and enhanced the transferrin content in hemolymph and iron deposition in the hepatopancreas when supplemented with 92.25 and 143.00 mg/kg of iron ( P < 0.05 ). Crabs fed the diet supplemented with 41.40 mg/kg of iron had the highest malondialdehyde content and the lowest superoxide dismutase activity in the hepatopancreas. Dietary 700.90 mg/kg of vitamin C supplementation also increased the acid phosphatase and bacteriolytic activity when diets were supplemented with 41.40 and 143.00 mg/kg of iron ( P < 0.05 ). In addition, crabs fed 143.00 mg/kg of iron and 700.90 mg/kg of vitamin C diet had the highest survival rate after the Aeromonas hydrophila challenge. This study demonstrates that dietary 700.90 mg/kg of vitamin C could promote iron absorption and utilization, thereby increasing the growth, immunity, and disease resistance of E. sinensis.

A novel l-rhamnose-binding lectin participates in defending against bacterial infection in zebrafish

l-rhamnose-binding lectin (RBL), which is a class of animal lectins independent of Ca2+, can specifically bind l-rhamnose or d-galactose. Although several lectins in zebrafish have been reported, their functional mechanisms have not been fully uncovered. In this study, we discovered a novel l-rhamnose binding lectin (DrRBL) and studied its innate immune function. The DrRBL protein contains only one carbohydrate-recognition domain (CRD), which includes two strictly conserved motifs, “YGR” and “DPC”. DrRBL was detected in all tested tissues and was present at high levels in the spleen, hepatopancreas and skin. After Aeromonas hydrophila challenge, the DrRBL mRNA level was significantly upregulated. Additionally, DrRBL was secreted into the extracellular matrix. Recombinant DrRBL (rDrRBL) could significantly inhibit the growth of gram-positive/negative bacteria, bind to several bacteria and cause obvious agglutination. The rDrRBL protein could combine with polysaccharides, such as PGN and LPS, rather than LTA. A more detailed study showed that rDrRBL could combine with monosaccharides, such as mannose, rhamnose and glucose, which are important components of PGN and LPS. However, rDrRBL could not bind to ribitol, which is an important component of LTA. The DrRBL deletion mutants, DrRBLΔ144-150 and DrRBLΔ198-200, were also constructed. DrRBLΔ144-150 (“ANYGRTD” deficient) showed weak bacterial inhibiting ability. However, DrRBLΔ198-200 (“DPC” deficient) showed weak agglutination ability. These results suggest that the “DPC” domain is important for agglutination. The conserved domain “ANYGRTD” is essential for inhibiting bacterial growth.

Feed for Nile tilapia broodstock and offspring supplemented with purified nucleotides boosts the juvenile’s health, growth, and the resistance face to transport and Aeromonas hydrophila challenges

This study evaluated the effects of feeding Nile tilapia broodstock and offspring with diets containing purified nucleotides on the health, growth, and resistance of juveniles subjected to different challenges. The broodstock received feed containing 0.0, 2.5, or 5.0 g nucleotides (NT) kg−1, and the offspring received feed containing 0.0, 2.5, 5.0, or 7.5 g NT kg−1. Growth and survival were evaluated on 45-days-old tilapia likewise the survival face to challenge by transport also was assessed. After, the growth, survival, hematological parameters, liver and plasma biochemical parameters were assessed on 105-days-old tilapia, likewise these parameters were also assessed on 105-days-old tilapia challenged by Aeromonas hydrophila. The growth of 45-day-old tilapia was affected by offspring feeding (p < 0.05), which those fed on 2.5 and 7.5 g NT kg−1 presented greater body weight. The survival and vigor of 45-days-old tilapia was affected (p < 0.05) by broodstock feeding (p < 0.05). The 45-days-old tilapia from breeders fed on 0.0 and 2.5 g NT kg−1 presented a higher survival, whereas 45-days-old tilapia from breeders fed on 2.5 and 5.0 g NT kg−1 presented a higher survival after transport. Broodstock and offspring fed with 2.5 g NT kg−1 produced larger 105-days-old tilapia (p < 0.05), which presented less liver lipoperoxidation (p < 0.05) and provided a greater biomass (p < 0.05). The 105-days-old tilapia fed with 2.5 g NT kg−1 that were from breeders fed on non-supplemented feed presented less hepatic superoxide dismutase activity after being challenged with bacteria. Broodstock and offspring both fed on 2.5 g NT kg−1 afforded 105-days-old tilapia with the lowest hepatic alanine aminotransferase and greater survival face to bacteria challenged. The dietary supplementation of purified nucleotides for both broodstock and offspring enabled early development and boosted the antioxidative status and liver integrity of 45 and 105-days-old Nile tilapia, resulting in better growth, health, and resistance when faced with different challenges.

CcGSDMEa functions the pore-formation in cytomembrane and the regulation on the secretion of IL-lβ in common carp (Cyprinus carpio haematopterus).

GSDME is the only direct executor of caspase-dependent pyroptosis in both canonical and non-canonical inflammasomes known to date in fish, and plays an important role in anti-bacterial infection and inflammatory response. In order to determine the regulation of GSDMEa on antibacterial infection in innate immune response, the CcGSDMEa gene in common carp (Cyprinus carpio haematopterus) was first identified and characterized, and then its function related to immune defense was investigated. Our results showed that the expressions of CcGSDMEa at the mRNA and protein levels were both significantly increased after Aeromonas hydrophila intraperitoneal infection at the early stage than that in the control group. We found that CcGSDMEa could be cleaved by inflammatory caspase (CcCaspase-1b) and apoptotic caspases (CcCaspase-3a/b and CcCaspase-7a/b). Interestingly, only the CcGSDMEa-NT (1-252 aa) displayed bactericidal activity to Escherichia coli and could punch holes in the membrane of HEK293T cells, whereas CcGSDMEa-FL (1-532 aa) and CcGSDMEa-CT (257-532 aa) showed no above activity and pore-forming ability. Overexpression of CcGSDMEa increased the secretion of CcIL-1β and the release of LDH, and could reduce the A. hydrophila burdens in fish. On the contrary, knockdown of CcGSDMEa reduced the secretion of CcIL-1β and the release of LDH, and could increase the A. hydrophila burdens in fish. Taken together, the elevated expression of CcGSDMEa was a positive immune response to A. hydrophila challenge in fish. CcGSDMEa could perform the pore-formation in cell membrane and the regulation on the secretion of IL-lβ, and further regulate the bacterial clearance in vivo. These results suggested that CcGSDMEa played an important role in immune defense against A. hydrophila and could provide a new insight into understanding the immune mechanism to resist pathogen invasion in teleost.

Identification and functional analysis of two drosophila mothers against decapentaplegic protein(Smad)genes and their involvement in immune responses in the red swamp crayfish Procambarus clarkii

Drosophila mothers against decapentaplegic proteins (Smads), the crucial signal transducers in activating downstream gene transcription through transforming growth factor beta (TGF-β) receptors, are the pleiotropic factors with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, whether Smads are involved in immune response in crustaceans remains unexplored. In the present study, the Smad3 and Smad4 were firstly identified and functionally characterized from the Red Swamp Crayfish Procambarus clarkii. The full-length cDNAs of pcSmad3 and pcSmad4 were 1, 670 bp and 3, 060 bp with 1, 326 bp and 1, 875 bp open reading frame (ORF), respectively. Real-time PCR analysis of the expression profiles demonstrated that pcSmad3 and pcSmad4 were predominantly expressed at in stomach, heart, and hemocytes. Notably, the expression levels of pcSmad3 and pcSmad4 both Aeromonas hydrophila and WSSV challenges were significantly altered, suggesting the involvement of pcSmad3 and pcSmad4 in innate immune responses. Knockdown of pcSmad3 and pcSmad4 in vivo dramatically activated the transcriptions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. The overexpression of pcSmad3 and pcSmad4 could significantly activate NF-κB signaling in HEK293T cells. Meanwhile, the clearance of bacteria was significantly reduced with knockdown of pcSmad3 and pcSmad4 in vivo. Results indicated that pcSmad3 and pcSmad4 played an immune-regulatory role in crayfish's innate immunity, which might pave the for a better understanding of the TGF-β superfamily members in crustacean.

Exploring the multimodal role of Cnicus benedictus extract in the modulation of growth, hematobiochemical, histopathological, antioxidative performance, and immune-related gene expression of Oreochromis niloticus challenged with Aeromonas hydrophila

This study explored the growth efficiency and the intracellular pathways by which Cnicus benedictus extract (CBE) acts. It investigated the antioxidant effects and efficacy of CBE as a fish supplement in attenuation of Aeromonas hydrophila in Oreochromis niloticus fish. Mono-sex Nile tilapia fish (n = 225) were randomly allocated to five groups in triplicate aquaria (n = 3 tanks per group, 15 fish per tank, with 120 L of water per tank) with a daily water exchange rate of 20%. After adaption for 2 weeks and body weight measuring, the experimental groups were fed isonitrogenous and isocaloric diets with different dosages of the ethanolic extract of C. benedictus for 10 weeks. The five groups were identified as the control group (CBE0.0), which was fed on the basal diet, while the second (CBE0.1), the third (CBE0.2), the fourth (CBE0.4), and the fifth (CBE0.6) groups were fed the basal diet supplemented with 0.1%, 0.2%, 0.4%, and 0.6% of C. benedictus extract, respectively. After the 10-week feeding trial was completed, the fish were inoculated with the PCR-identified pathogenic A. hydrophila in a challenge trial which lasted 15 days. A. hydrophila, one of the septicemic bacteria, causes severe economic losses, high mortality rates, and hemorrhages in Nile tilapia and other cultured freshwater fishes worldwide. The CBE was found to significantly increase the body mass, weight gain, and the specific growth rate, as well as the protein efficiency ratio of the fish. Increased survival percentage, accompanied by post challenge lymphocytosis with decreased liver enzyme levels, increased total protein, and improved kidney function markers were also seen. Additionally, CBE supplementation showed significant increases in phagocytic activity, phagocytic index, and lysosomal activity post challenge, accompanied by increases in antioxidant activity and the mRNA expression of cytokines genes hsp70 and tlr7 mRNA. The desirable effects of CBE treatment were confirmed by a histopathological examination of the height of intestinal villi and enterocytes lining the middle intestine and increases in the size of liver cells. We conclude that CBE increases the growth performance and modulates the antioxidant, inflammatory, stress, and immune-related genes in Nile tilapia. Moreover, the dietary inclusion of 0.42–0.47% CBE showed a better protective effect with the A. hydrophila challenge.

Integrated analysis of gene expressions and metabolite features unravel immunometabolic interplay in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) infected with Aeromonas hydrophila

Aeromonas hydrophila is one of important pathogenic bacteria that can imperil fish survival. Current study suggested that hybrid crucian carp (WR) receiving acute A. hydrophila challenge exerted a molecular regulation linking peroxisomal fatty acid β-oxidation to immune response. A. hydrophila infection may alleviate antioxidant status along with the significant alternation of immune-, apoptosis- and metabolic-related gene expressions in kidney, whereas most portions of differential metabolites (DMs) were enriched in steroid derivatives and phospholipids. In addition, mevalonate (MVA) pathway and phospholipid hydrolysis were identified as crucial cellular processes in WR after A. hydrophila challenge. These results may provide an insight into immunometabolic strategy against A. hydrophila infection.

Dietary chitosan moderates the growth rate, antioxidant activity, immunity, intestinal morphology and resistance against Aeromonas hydrophila of juvenile hybrid sturgeon (Acipenser baerii♀ × Acipenser schrenckii♂)

This study investigated the effects of dietary chitosan on growth, antioxidant, immunity, intestinal morphology and resistance against Aeromonas hydrophila of hybrid sturgeon (Acipenser baerii♀ × Acipenser schrenckii♂). Sturgeons (18.18 ± 0.08 g) were randomly divided into four groups, fed with chitosan-supplemented diets for 8 weeks and then infected with A. hydrophila. The results showed significant differences of body weight gain, specific growth rate and feed conversion ratio in sturgeon fed chitosan and control diets. The oral administration of chitosan significantly increased the acid phosphatase, alkaline phosphatase, lysozyme, myeloperoxidase, superoxide dismutase, glutathione peroxidase and catalase activities, as well as the complement 3 and 4 contents and disease resistance against A. hydrophila. Moreover, enhancement of muscular thickness and goblet cells in mid intestine and increase of muscular thickness and villus height in spiral valve were observed in the chitosan supplemented groups. In addition, dietary chitosan-supplemented diets mitigated the changes of antioxidant and immune activity induced by A. hydrophila challenge, as well as prevented fish from bacterial invasion. The optimal dose was 3.00 g chitosan/kg diet for hybrid sturgeon.

Metabolite features of serum and intestinal microbiota response of largemouth bass (Micropterus salmoides) after Aeromonas hydrophila challenge

The enteric morphology, enteric microbiota structure and serum metabolomics of M. salmoides before and after infected by A. hydrophila were analysed to explore the pathogenic mechanism of A. hydrophila infection in M. salmoides. The results revealed that, after the infection of A. hydrophila, the villus boundary of largemouth bass became less obvious; the relative abundance of Proteobacteria and decreasing relative abundance of Tenericutes were increasing; genera relative abundance of putatively beneficial bacteria (Mycoplasma) were decreasing, whereas the genus Aeromonas increased after infection; serum metabolomic analysis showed that infection with A. hydrophila caused disorder to the metabolic processes of largemouth bass, particularly amino acid metabolism, and caused inflammation; several potential pathogen infection-related and significantly differential intestinal microbiota-related metabolite markers were identified, such as 6-hydroxy-5-methoxyindole glucuronide, zalcitabine, bilirubin, aciclovir. This study may provide new insights into the potential association between enteric microbiota and serum metabolism and the pathogenic mechanism of M. salmoides infected by A. hydrophila, providing a scientific basis for disease control in largemouth bass breeding.

Protective efficacy evaluation of immunogenic protein AHA_3793 of Aeromonas hydrophila as vaccine candidate for largemouth bass Micropterus salmoides.

Aeromonas hydrophila is a Gram-negative pathogen that can infect various fish, including largemouth bass (Micropterus salmoides), which have caused huge economic losses. In present study, largemouth bass anti-A. hydrophila antibodies were produced, then a highly immunogenic outer membrane proteins, AHA_3793, was identified by combined western blotting and mass spectrometry analysis. Moreover, AHA_3793 was expressed, and its immunogenicity was further verified by western blotting. Subsequently, the protective efficacy of AHA_3793 were evaluated in largemouth bass. The results showed that rAHA_3793 could produce a relative percentage survival (RPS) of 61.76% for largemouth bass against A. hydrophila challenge. ELISA analysis showed the specific serum antibodies of largemouth bass against rAHA_3793 and A. hydrophila in vaccinated group in weeks 4 and 5 after immunization were significantly higher than those in control group, which suggested that rAHA_3793 induced production of specific serum antibodies against rAHA_3793 and A. hydrophila. The qRT-PCR analysis showed that expressions of CD4-2 and MHC IIα were also significantly up-regulated after immunization. These results collectively demonstrated that rAHA_3793 could induce a strong humoral immune response of largemouth bass, and then produce high immune protection effects against A. hydrophila infection.Electronic supplementary materialSupplementary material (Supplementary Figs.S1–S2) is available in the online version of this article at 10.1007/s00343-022-1326-8.

A multispecies probiotic modulates growth, digestive enzymes, immunity, hepatic antioxidant activity, and disease resistance of Pangasianodon hypophthalmus fingerlings

An eight-week trial was designed by feeding Pangasianodon hypophthalmus fingerlings on diets supplemented with a multispecies probiotic comprised of three probiotic bacteria (Bacillus subtilis, Lactobacillus plantarum, and Enterococcus faecium) in combination with a probiotic yeast (Saccharomyces cerevisiae). The effects were observed regarding growth, body composition, digestive enzymes, liver function enzymes, serum metabolites, immunological responses, hepatic antioxidant capacity, and resistance to the challenge with Aeromonas hydrophila. Fish (9.54 ± 0.50 g) were allocated into four triplicate groups and were fed on formulated pelleted diets enriched with varying levels of the probiotic as 0.0 (reference or CONT), 0.5, 1.0, and 1.5 g per 1 kg feed. After the feeding trial ended, a significant augmentation of the growth performance in terms of the fish's final weight, weight gain and specific growth rate were observed in the probiotic-enriched groups in comparison to the CONT group. There were also improvements in the feed intake and feed conversion ratio. Nevertheless, no variations were noticed in whole-body analysis, liver enzymes (AST, ALT, and ALP), and serum metabolites (blood urea nitrogen, uric acid, and creatinine) among the experimental groups. Meanwhile, digestive enzymes (lipase, amylase, and protease) and enzymatic antioxidant activities (CAT, SOD, and GPx) were increased in all probiotic-supplied groups in comparison with the CONT group. On the other side, hepatic malondialdehyde concentrations declined in probiotic-supplemented groups in relation to the CONT group. An improved immunological response (serum lysozyme activity and total immunoglobulins) was aligned with the higher cumulative survival of P. hypophthalmus in response to the pathogenic A. hydrophila challenge. Taken together, the enhancement of the growth, antioxidant activity, and immunity, aligned with increased resistance to A. hydrophila, can suggest the potentiality of dietary supplementation with a multispecies probiotic composed of probiotic bacteria and yeast to improve the functionality and effectuality of aquadiets prepared for this valuable fish species.

Class a scavenger receptor-A5 gene in Cirrhinus mrigala: Cloning, characterisation and expression patterns in response to bacterial infection

• Cirrhinus mrigala scavenger receptor (CmSRA5) was identified and characterized. • CmSRA5 showed strong binding ability with the lipid A component of bacterial LPS. • mRNA expression of CmSRA5 in the early developmental stages of C. mrigala was reported. • CmSRA5 mRNA expression levels in the different tissues were significantly induced in response to the A. hydrophila challenge.

The Effects of Dietary Thyme Oil (Thymus vulgaris) Essential Oils for Common Carp (Cyprinus carpio): Growth Performance, Digestive Enzyme Activity, Antioxidant Defense, Tissue and Mucus Immune Parameters, and Resistance against Aeromonas hydrophila.

This study evaluated the effects of dietary supplementation of thyme (Thymus vulgaris) essential oil (TVO) on growth performance, digestive enzymes, biochemical parameters, hematological indices, liver enzymes, and pathogen resistance in common carp (Cyprinus carpio). Triplicate groups of fish (15.36 ± 0.10 g) were fed daily with diets supplemented with TVO at 0, 0.5, 1, and 2 percent for 60 days then challenged with Aeromonas hydrophila. The results determined that supplementation of thyme resulted in significantly higher final body weights (FBW) and lower feed conversion ratios (FCR). Furthermore, no mortality was observed in the thyme-supplemented treatments. Regression analysis showed that fish growth parameters were polynomially related to dietary TVO levels. The optimum dietary TVO level, based upon the varied growth parameters, was 1.344 to 1.436%. Digestive enzymes activity, including amylase and protease, significantly increased in fish fed the supplemented diets. The thyme-supplemented diets also significantly increased the biochemical parameters, including total protein, albumin, and acid phosphatase (ACP), compared to the control group. We also observed significant increases in hematological indices, including red blood cells (RBC), white blood cells (WBC), hematocrit (Hct), and hemoglobin (Hb) in common carp fed diets containing thyme oil (P < 0.05). Liver enzymes activity including alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) was also reduced (P < 0.05). Immune parameters, including total protein and total immunoglobulin (total Ig) levels, alternative complement pathway hemolytic (ACH50), lysozyme, protease, and ALP in the skin mucus, and lysozyme, total Ig, and ACH50 in the intestine were higher (P < 0.05) in TVO-supplemented fish. Catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) in the liver were also elevated (P < 0.05) in TVO administered groups. Lastly, thyme-supplementation resulted in higher survival rates after the A. hydrophila challenge compared to the control (P < 0.05). In conclusion, dietary inclusion of thyme oil (1 and 2%) effectively improved fish growth, immune systems, and resistance to A. hydrophila.

MEK homologue is involved in immune response by regulating antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis

MEK activates the phosphorylation of downstream molecules involved in various immune responses. In this study, an MEK homologue gene in Chinese mitten crab Eriocheir sinensis (designated as EsMEK) was investigated. EsMEK mRNA was constitutively expressed in all tissues with higher expression in hepatopancreas, hemocytes, and gills. EsMEK protein was mainly localized in the cytoplasm. Lipopolysaccharide (LPS) and Aeromonas hydrophila challenge significantly increased the mRNA levels of EsMEK in hemocytes. In addition, the mRNA expression level of some antimicrobial peptides (AMPs), including EsWAP, EsDWD1, and EsALF decreased significantly due to the inhibition of EsMEK by specific dsRNA in LPS-challenged crabs. Downstream pathway analysis revealed that the phosphorylation of EsERK decreased prominently after EsMEK inhibition. These results suggested that EsMEK played an important role in regulating the expression of antimicrobial peptides in E. sinensis through MEK-ERK pathway.

Optimal DL-Methionyl-DL-Methionine Supplementation Improved Intestinal Physical Barrier Function by Changing Antioxidant Capacity, Apoptosis and Tight Junction Proteins in the Intestine of Juvenile Grass Carp (Ctenopharyngodon idella).

The present study was a part of a larger research project that aimed to investigate the effects of Met-Met supplementation on fish growth and intestinal health. This study mainly focused on the relationship between dietary Met-Met and intestinal physical barrier function in fish. Seven iso-nitrogenous diets supplemented with 2.50 g/kg DL-methionine (DL-Met) and six graded levels of Met-Met (0.00, 0.79, 1.44, 1.84, 2.22, and 2.85 g/kg) were used to feed juvenile grass carp for 10 weeks, after which a 14-day Aeromonas hydrophila challenge test was performed. The results indicated that optimum levels of Met-Met decreased intestinal oxidative damage, probably by increasing total antioxidant capacity, and the activity and gene expression levels of several antioxidant enzymes, which were closely related to the changed Nrf2/Keap1 signaling. Meanwhile, optimum levels of Met-Met decreased intestinal apoptosis and improved the intestinal tight junction, as evident by the downregulated mRNA levels of initiator and executioner caspases; the pro-apoptotic-related proteins FasL, Apaf-1, and Bax; and upregulated mRNA levels of the anti-apoptotic proteins Bcl-2, Mcl-1b, and IAP and the TJ proteins claudins, occludin, and ZOs. Furthermore, the positive effects of Met-Met on improving intestinal physical barrier function were superior to those of DL-Met in fish. These findings showed that optimal Met-Met supplementation improved intestinal physical barrier function, probably by changing antioxidant capacity, apoptosis, and tight junction proteins in fish.

A different transcriptional landscape sheds light on Russian sturgeon (Acipenser gueldenstaedtii) mechanisms to cope with bacterial infection and chronic heat stress

Sturgeons are chondrostean fish of high economic value and critically endangered due to anthropogenic activities, which has led to sturgeon aquaculture development. Russian sturgeon (Acipenser gueldenstaedtii), the second most important species reared for caviar, is successfully farmed in subtropical countries, including Uruguay. However, during the Uruguayan summer, sturgeons face intolerable warmer temperatures that weaken their defences and favour infections by opportunistic pathogens, increasing fish mortality and farm economic losses. Since innate immunity is paramount in fish, for which the liver plays a key role, we used deep RNA sequencing to analyse differentially expressed genes in the liver of Russian sturgeons exposed to chronic heat stress and challenged with Aeromonas hydrophila. We assembled 149.615 unigenes in the Russian sturgeon liver transcriptome and found that metabolism and immune defence pathways are among the top five biological processes taking place in the liver. Chronic heat stress provoked profound effects on liver biological functions, up-regulating genes related to protein folding, heat shock response and lipid and protein metabolism to meet energy demands for coping with heat stress. Besides, long-term exposure to heat stress led to cell damage triggering liver inflammation and diminishing liver ability to mount an innate response to A. hydrophila challenge. Accordingly, the reprogramming of liver metabolism over an extended period had detrimental effects on fish health, resulting in weight loss and mortality, with the latter increasing after A. hydrophila challenge. To our knowledge, this is the first transcriptomic study describing how chronic heat-stressed sturgeons respond to a bacterial challenge, suggesting that liver metabolism alterations have a negative impact on the innate anti-bacterial response.

An Evaluation of Yeast Culture Supplementation in the Diet of Pseudobagrus ussuriensis: Growth, Antioxidant Activity, Nonspecific Immunity, and Disease Resistance to Aeromonas hydrophila

An 8-week feeding trial was conducted to evaluate the effects of dietary yeast culture (YC) supplementation on growth performance, antioxidant activity, nonspecific immunity, and disease resistance of Pseudobagrus ussuriensis (average initial weight6.01±0.01 g). Four isonitrogenous and isolipidic diets were formulated to contain 0 (Y0), 10 (Y1), 20 (Y2), and 30 (Y3) g/kg YC, respectively. After the feeding experiment, the challenge test of injecting Aeromonas hydrophila was executed. Results showed that appropriate YC supplementation level in the diet could improve growth performance, digestive enzyme activities, nonspecific immunity capacity, antioxidant capacity, and disease resistance of P. ussuriensis. And the highest weight gain, feed intake, specific growth rate, and IGF-1 gene expression level were observed in fish fed the Y2 diet. The activities of protease and amylase in intestine in fish fed the Y2 diet were enhanced compared with that in fish fed the Y0 diet significantly (P&lt;0.05). Simultaneously, fish fed the Y2 diet had significantly higher serum lysozyme activity and significantly lower serum alanine amino transferase activity (P&lt;0.05). Dietary 20 g/kg YC supplementation increased the activity of catalase and total antioxidant capacity in liver and reduced malondialdehyde content in the liver and intestine of P. ussuriensis significantly (P&lt;0.05). Fish fed the Y2 diet had the highest disease resistance under the condition of A. hydrophila challenge (P&lt;0.05). The quadratic regression analysis based on weight gain against dietary YC levels indicated that the appropriate dietary YC supplementation level is 13.4 g/kg diet.

Evaluation of dietary Vitamin C requirement of Hypophthalmichthys molitrix fingerlings and its effects on growth, haematology and serum enzyme activities

Vitamin C (VC) is a necessary nutrient for normal fish physiology and immunity. Therefore, A 60-day feeding trial was designed to evaluate the dietary VC requirements of silver carp (Hypophthalmichthys molitrix). Six isocaloric and isonitrogenous diets were formulated by incorporation of VC at 0, 50, 100, 200, 400 and 800 mg kg−1 levels and the analysed VC content in diet was 5.7, 51.1, 95.3, 191.6, 370.7 and 757.5 mg kg−1 respectively. At the trial termination, VC supplementation significantly enhanced the growth and feed utilization. The highest values for whole body crude protein were recorded at 95.3 and 191.6 mg kg−1 of VC. In comparison to the basal diet, VC supplementation improved (p < 0.05) the whole body crude fat. Dietary VC had a dose-dependent effect on the white blood cell count and haematocrit. A significant increase in the alkaline phosphatase activity was recorded for fish fed 5.7 to 95.3 mg kg−1 of VC, attaining a constant value afterwards (95.3–757.5 mg kg−1 of VC). In comparison, dietary VC decreased (p < 0.05) the aspartate aminotransferase and alanine aminotransferase activities. Similarly, the glucose content also decreased significantly from 5.7 to 95.3 mg kg−1 of VC and became constant thereafter. Dietary VC supplementation significantly affected the antioxidant enzyme activities. The liver VC content showed a dose-dependent relation, where the VC content increased with the increase in VC supplementation reaching the highest value at 757.5 mg kg−1. Moreover, VC-fed fish showed an increased (p < 0.05) survival against Aeromonas hydrophila challenge. On the basis of broken-line regression, the optimum dietary VC requirement of H. molitrix for weight gain% and liver VC content is 61.31 and 241.13 mg kg−1 respectively.

Impact of dandelion polysaccharides on growth and immunity response in common carp Cyprinus carpio

Natural products have gained considerable attention for improving fish growth performance and immunity to enhance disease resistance. This study evaluated the effect of dandelion polysaccharides (DP) on skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge in the Common carp Cyprinus carpio. Diets containing four different concentrations of DP (g Kg−1):0 g [basal diet], 0.5 g [D1], 1.5 g [D2], 2.5 g [D3], and 4.0 g [D4] were fed to the carp (average weight: 13.92 ± 0.83 g) for eight weeks. Growth parameters were analyzed four and eight weeks after feeding. Immunological, hematological, and antioxidant parameters were examined eight weeks post-feeding. Growth performance was significantly higher on D3, with a final weight gain of 71.48 ± 1.57 g and a specific growth rate of 3.06 ± 0.12. Among hematological parameters examined, erythrocyte, hematocrit, and mean corpuscular volume (MCV) levels were significantly higher in D3. Skin mucosal immune parameters, such as lysozyme (31.04 ± 1.02 Unit mL−1), alkaline phosphatase (122.6 ± 3.8 IU L-1), and protein level (10.6 ± 0.74 mg mL−1) were significantly higher in D3, while peroxidase activity was higher in D4. Furthermore, SOD activity was higher in D2–D3, whereas catalase activity was higher in D2–D4 (P < 0.05) than in the control. Malondialdehyde level decreased significantly in D3 (5.43 ± 0.36 nmol mL−1); whereas, serum ALT and AST levels were significantly lower on D2-D4. Intestinal tight-junction-related genes ZO-1 and Claudin 7 were significantly higher in the DP-fed groups; however, DP had no significant effect on claudin 3. Occludin expression was higher (p < 0.05) on D3 only. Pro-inflammatory cytokines (IL-1β and TNF-α) and IFN–γ strongly upregulated in the head kidney at D3. Conversely, the expression of the anti-inflammatory cytokine interleukin-10, HSP70, and TOR were considerably downregulated in D3. Fish from D3 exhibited markedly higher relative post-challenge survival (66.67%) against Aeromonas hydrophila challenge. The results of the present study suggest that dietary supplements of DP at 2.5 g kg−1 can significantly improve the growth performance, skin mucosal, and serum antioxidant parameters, and strengthen the immunity of C. carpio. Therefore, DP is a promising food additive for carp aquaculture.