Hydrophila Challenge Research Articles

Novel pectin isolated from Spirulina maxima enhances the disease resistance and immune responses in zebrafish against Edwardsiella piscicida and Aeromonas hydrophila

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 μg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 μg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1β: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1β) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.

Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.

Previous studies have shown that ATP synthase α subunit (ATP5A1) plays multiple roles, but our understanding of its biologic functions remains poor and incomprehensive. Here, we clearly demonstrated that zebrafish ATP5A1 was a newly characterized lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs and embryos of zebrafish. Zebrafish ATP5A1 acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, but also as an effector molecule, capable of inhibiting the growth of both gram-positive and -negative bacteria. ATP5A1 could disrupt the bacterial membranes by a combined action of membrane depolarization and permeabilization. We also found that the N-terminal 65 residues were critical for the antibacterial activity of zebrafish ATP5A1. In particular, we showed that microinjection of exogenous recombinant (r)ATP5A1 into early embryos could promote their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was markedly reduced by the coinjection of anti-ATP5A1 antibody or by the knockdown with morpholino for atp5a1 but not by the coinjection of anti-actin antibody. Moreover, each egg/embryo contains a sufficient amount of ATP5A1 in vivo to kill A. hydrophila. Furthermore, the N-terminal 65 residues 1-65 of ATP5A1 α subunit (rA1-65) with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the N-terminal 69 residues 66-134 (rA66-134) or C-terminal residues 135-551 (rA135-551) of ATP5A1 α subunit without in vitro antibacterial activity did not. Finally, we showed that the antibacterial activity of the N-terminal 65 residues of ATP5A1 α subunit was conserved throughout animal evolution. Collectively, these results indicate that ATP5A1 is a novel maternal immunocompetent factor that can protect the early embryos of zebrafish from bacterial infection. This work also provides a new viewpoint for understanding the biologic roles of ATP5A1, which is ubiquitously present in animals.-Ni, S., Zhou, Y., Chen, Y., Du, X., Zhang, S. Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.

First identification and characterization of a triple WAP domain containing protein in Procambarus clarkii provides new insights into the classification and evolution of WAP proteins in crustacean

Whey acidic protein domain (WAPD) is a usual motif in crustaceans, and is found mainly in the immune-related proteins. In the present study, a protein containing three tandem WAPDs was identified in red swamp crayfish Procambarus clarkii and designated as PcTWD. This is the first report of a protein of such domain architecture in crustaceans. Introducing the WAPDs of PcTWD into phylogenetic analysis led to the classification of crustacean WAP proteins into classical crustins and proteins containing solely WAPDs. PcTWD was widely expressed in multiple tissues, including hemocytes, gills, hepatopancreas, heart, stomach and intestine. Its expression could be significantly induced by Staphylococcus aureus or Aeromonas hydrophila challenge. Knockdown PcTWD expression by RNAi suppressed host resistance against A. hydrophila, while exogenous recombinant PcTWD could enhance the host immunity. The three WAPDs showed a labor division. The first two domains were responsible for the protease inhibitory activity, and the third domain contributed to the antimicrobial activity. Thus PcTWD was found as an important protein in crayfish antibacterial immunity.

Effects of dietary heat-killed Pseudomonas aeruginosa strain VSG2 on immune functions, antioxidant efficacy, and disease resistance in Cyprinus carpio

A probiotic strain of Pseudomonas aeruginosa VSG2 was previously isolated from the gut of Indian major carp Labeo rohita. But, its efficacy as a heat-inactivated probiotic on the immune response of fish remains unexplored. Thus, present study investigated the effect of heat-killed P. aeruginosa VSG2 (HK-PA) supplementation on the immune function, digestive enzymatic activities, and antioxidant status of Cyprinus carpio. Moreover, the effect of HK-PA supplementation on the susceptibility of C. carpio to Aeromonas hydrophila challenge was also evaluated. To do so, diets containing five different concentrations of HK-PA (mg kg−1) (0 [basal diet], 10 [PA10], 20 [PA20], 30 [PA30], and 40 mg kg−1 [PA40]) were fed to fish for 8 weeks. The results revealed that immune-related factors, such as lysozyme, protein level, alkaline phosphatase, and alternative complement pathway levels increased in fish fed with HK-PA, but differences were significant in PA20–PA40 compared to the control. The immunoglobulin level was higher (p < .05) in PA30, compared to the control. Superoxide dismutase (SOD), glutathione, glutathione peroxidase (GPx), and myeloperoxidase levels significantly increased in serum or skin mucus of fish fed with HK-PA. Accordingly, malondialdehyde levels decreased in fish fed with HK-PA. Among intestinal enzymatic activities, protease activity was higher in the HK-PA fed groups compared to the control. Moreover, mRNA expression of antioxidant genes (SOD, GPx, and catalase) significantly increased in the liver of PA20–PA40. Fish in the PA30 exhibited the highest relative post-challenge survival (71.54 ± 5.37%) against A. hydrophila infection. Overall, dietary administration of HK-PA at 30 mg kg−1 (PA30) for 8 weeks improved (p < .05) the skin mucosal and serum immune parameters, strengthened antioxidant capacity, enhanced intestinal enzymatic activities, and increased the resistance against a pathogen challenge. The heat-killed P. aeruginosa VSG2 represents a promising dietary supplement for carps in aquaculture.

Identification and characterization of five Nk-lysins from Pseudocrossocheilus bamaensis and their diverse expression patterns in response to bacterial infection

Nk-lysin is an effector protein of cytotoxic T lymphocytes and natural killer cells. It is known to possess anti-bacterial, anti-fungal, anti-viral, and anti-tumor activity. Here we describe five Nk-lysin genes (PbNkla, PbNklb, PbNklc, PbNkld, and PbNkle) from Pseudocrossocheilus bamaensis, a rare indigenous species distributed in Guangxi, China. The open reading frames (ORFs) consisted of 426 (PbNkla), 435 (PbNklb), 369 (PbNklc), 366 (PbNkld), and 339 (PbNkle) bp nucleic acids. The surfactant-associated protein B (SapB) domain and six conserved cysteine residues were identified in each PbNkl gene. Phylogenetic analysis revealed similar results to homology comparison that PbNkla and PbNklb consist of five exons and four introns and grouped together, whereas PbNklc and PbNkld each contain four exons and three introns and formed a separate clade. PbNkle had three exons and two introns and formed an independent clade separate from the four other PbNkls. qPCR analysis demonstrated that PbNkla, PbNklc, PbNkld, and PbNkle were ubiquitously expressed in all tissues examined, whereas PbNklb was expressed only after bacterial infection. Aeromonas hydrophila challenge significantly up- and down-regulated PbNkls at different time points post-injection and in different immune-related tissues. These results suggested that PbNkls were conserved immune molecules that may be involved in the immune response to pathogen invasion.

Dietary addition of rutin impairs inflammatory response and protects muscle of silver catfish (Rhamdia quelen) from apoptosis and oxidative stress in Aeromonas hydrophila-induced infection

This research aimed to assess the influence of dietary addition of rutin on inflammation, apoptosis and antioxidative responses in muscle of silver catfish (Rhamdia quelen) challenged with Aeromonas hydrophila (A. hydrophila). Fish were split into four groups as follows: control, 0.15% rutin, A. hydrophila, 0.15% rutin + A. hydrophila. After 2 weeks of feeding with standard or rutin diets, fish were challenged or not with A. hydrophila for 1 week. Rutin-added diet abrogates A. hydrophila induced-hemorrhage and inflammatory infiltration. It decreases A. hydrophila induced-apoptosis through decreasing the ratio of Bax to Bcl-2 and increasing phospho-Akt to Akt ratio. It diminishes the A. hydrophila induced-rise in nitric oxide and superoxide anion levels and reestablishes superoxide dismutase activity as well. Although such diet is unable to recover the levels of reduced glutathione (GSH), cysteine and glutamate cysteine ligase, which are depleted as a result of A. hydrophila infection, it diminishes the oxidized glutathione (GSSG) content, thus decreasing GSSG to GSH ratio. It increases the levels of cysteine residues of proteins and diminishes those of thiol-protein mixed disulfides, which were changed after A. hydrophila challenge. Finally, it reduces A. hydrophila induced-lipid peroxidation, markedly elevates ascorbic acid and thus reestablishes total antioxidant capacity, whose levels were decreased after A. hydrophila challenge. In conclusion, the dietary addition of rutin at 0.15% impairs A. hydrophila-induced inflammatory response, inhibits A. hydrophila-induced apoptosis and promotes cell survival. It also reduces the A. hydrophila-induced oxidative stress and stimulates the antioxidative responses in muscle of A. hydrophila-infected silver catfish.

Ascorbic acid enhanced the growth performance, oxidative status, and resistance to Aeromonas hydrophila challenge of juvenile grass carp (Ctenopharyngodon idella)

This study was conducted to examine the dietary ascorbic acid requirement and effects on growth performance, oxidative status, and disease resistance in juvenile grass carp (Ctenopharyngodon idella). Triplicate groups of 30 fish weighting 3.39 ± 0.03 g were fed with diets containing graded levels of ascorbic acid (18.9, 71.8, 118.7, 169.9, 321.1, and 618.5 mg kg−1) in a recirculating rearing system for 8 weeks. No significant differences in growth performance among all groups were observed (P > 0.05). Diet with 321.1 mg kg−1 ascorbic acid significantly increased the feed intake (P < 0.05). Meanwhile, the expression of liver peroxisome proliferator-activated receptors-γ and uncoupling protein 2 were significantly declined (P < 0.05). Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila (2.4 × 105 CFU cells mL−1 per fish) and fish fed with ascorbic acid shown a better liver oxidation status (e.g., superoxide dismutase and glutathione peroxidase) than the control diet (P < 0.05). The optimum dietary ascorbic acid concentration was about 155 mg kg−1 basing on the oxidation status in liver. Nevertheless, the broken-line model indicated that the optimal level for juvenile grass carp is about 310 mg kg−1.

Subfunctionalization and evolution of liver-expressed antimicrobial peptide 2 (LEAP2) isoform genes in Siberian sturgeon (Acipenser baerii), a primitive chondrostean fish species

Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species.

Molecular Characterization and Heterologous Production of the Bacteriocin Peocin, a DNA Starvation/Stationary Phase Protection Protein, from Paenibacillus ehimensis NPUST1.

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.

Global characterization and expression analysis of interferon regulatory factors in response to Aeromonas hydrophila challenge in Chinese soft-shelled turtle (Pelodiscus sinensis)

Interferon regulatory factors (IRFs) were originally identified as transcriptional regulators of type I interferon (IFN) expression. Recent studies have widely identified the roles of IRFs as central mediators in immune defence against pathogen infection. However, the functional roles and expression profiles of IRFs are still unclear in Chinese soft-shelled turtle (Pelodiscus sinensis). In this study, eight members of the PsIRF family were identified in P. sinensis through a genome-wide search. These PsIRF genes contained the conserved domains of this group of proteins, including the N-terminal DNA-binding domain and C-terminal IRF-associated domain. Phylogenetic analyses among IRF homologs showed that the PsIRFs shared the closest phylogenetic relationships with IRFs of other turtle species. Further molecular evolutionary analyses revealed evolutionary conservation of the PsIRF genes. Moreover, expression profiling demonstrated that eight PsIRF genes exhibited constitutive expression in different tissues of P. sinensis. Several genes, such as PsIRF1, PsIRF2 and PsIRF4, showed predominant expression in the spleen and were significantly upregulated upon Aeromonas hydrophila infection. Remarkably, PsIRF1, PsIRF2 and PsIRF4 exhibited rapid increases in their protein expression levels post-infection and were mainly expressed in the splenic red pulp according to immunohistochemistry analysis. These results provide rich resources for further exploration of the roles of PsIRFs in immune regulation in P. sinensis and other turtles.

Effects of bioactive substance from turmeric on growth, skin mucosal immunity and antioxidant factors in common carp, Cyprinus carpio

Present study evaluated the effects of curcumin, the principal curcuminoid of turmeric, on Cyprinus carpio growth, skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge. Diets containing four various concentrations of curcumin (g Kg−1): 0 g [basal diet], 5 g [T5], 10 g [T10], and 15 g [T15] were fed to the carp (average weight: 16.37 g) for 8 weeks. Growth parameters were analysed at 4 and 8 weeks post-feeding. Skin mucosal immune responses and expression were examined in 8 weeks post-feeding. Growth performance was significantly higher in T10 and T15, with final weight gain of 102.26 ± 2.31 g and specific growth rate of 3.24 ± 0.37, respectively. The lowest feed conversion ratio (2.35 ± 0.16) was recorded in T15 than in the control (P < 0.05). Among the skin mucosal immune parameters examined, lysozyme (36.8 ± 4.03 U mL−1), total immunoglobulin (6.74 ± 0.5 mg mL−1), protein level (18.7 ± 1.62 mg mL−1), alkaline phosphatase (96.37 ± 6.3 IU L−1), and protease activity (9.47 ± 0.82%) were significantly higher in T15, while the peroxidase activity was higher in T10 (10.24 ± 0.9 U mg−1 protein). Further, lysozyme, superoxide dismutase (SOD) and catalase (CAT) activities were measured in serum and found to be higher in T10 or T15 than in the control (P < 0.05). However, malondialdehyde level decreased significantly in T10 and T15. Furthermore, antioxidant genes (SOD, CAT, nuclear factor erythroid 2-related factor 2) and anti-inflammatory cytokine Interleukin-10 were upregulated in the head kidney, intestine, and hepatopancreas of fish in T10 and T15. Conversely, expression of pro-inflammatory cytokines (IL-1β, tumour necrosis factor-alpha), signalling molecule NF-κBp65 were down-regulated in the tested tissues of T10 and T15. Expression of Toll-like receptor 22 (TLR22) was down regulated in head-kidney and intestine of T15. Fish from T15 exhibited significantly higher relative post-challenge survival (69.70%) against Aeromonas hydrophila challenge. Results of the present study suggest that dietary supplements of curcumin at 15 g Kg−1can significantly improve the growth performance, skin mucosal and serum antioxidant parameters, and strengthen the immunity of C. carpio. Therefore, curcumin represents a promising food additive for carps in aquaculture.

Hepcidin gene of Indian major carp, Labeo rohita: Molecular, structural and functional characterization, and antibacterial activity of recombinant hepcidin

Hepcidin, an antimicrobial peptide produced in the liver, also regulates iron balance and recycling in vertebrates. To study the antibacterial activity of Labeo rohita hepcidin (LrHep), full-length cDNA of LrHep was cloned. The 800 bp amplicon contained an open reading frame of 276 bp, encoding 91 amino acids including a signal peptide of 24 amino acid residues. The 3D structure and domain architecture of LrHep were predicted. Phylogenetic analysis of the LrHep sequence showed that it formed a sister group with another carp, Sinocyclocheilus rhinocerous. LrHep has eight cysteine residues at C73, C76, C77, C79, C80, C85, C88 and C89 positions conserved throughout evolution. Besides cysteine residues, like in all teleosts, A24, P26, E34 and R63 were also found to be conserved in LrHep. Expression analysis of LrHep using 12 different tissues of naïve rohu juveniles revealed that its expression is being the highest in the liver followed by anterior kidney. To study the role of LrHep during disease, its expression kinetics was observed in three infection models (Aeromonas hydrophila, poly I:C, and Argulus siamensis, representatives of bacterial, viral, and parasitic infections, respectively). Significant up-regulation of LrHep was observed in the liver in all the three experimental groups (at 12 h with A. hydrophila infection, 3 h with poly I:C stimulation and 24 h with A. siamensis infection). Significant up-regulation in anterior kidney was observed at 3 and 6 h only in A. hydrophila infected fish. The antimicrobial activity of LrHep was evaluated both in vitro and in vivo by producing recombinant hepcidin (approximately 11 kDa) of rohu (rLrHep) in Escherichia coli. The recombinant LrHep inhibited growth of the bacteria, Edwardsiella tarda and A. hydrophila, in vitro at concentrations of 50 and 100 μg (4.54 and 9.09 μM), respectively, at 6 h post-incubation. Administration of rLrHep to rohu juveniles rendered protection against A. hydrophila challenge with a relative percent survival (RPS) of 73%. Overall, the findings of this study suggest that owing to its potential antimicrobial activity, LrHep could be an effective immunomodulator providing protection against A. hydrophila infection.

Heritability of immunity traits and disease resistance of bighead catfish, Clarias macrocephalus Günther, 1864

Disease outbreak is a major obstruction for intensive aquaculture worldwide. One of the promising solutions is genetic improvement by selective breeding, providing that a sufficient proportion of additive genetic variance (measured by heritability-h2) of disease resistance traits exists. In addition, immunity traits are of interest as potential indirect targeted traits for disease resistance. In this study, the genetic parameters of resistance to Aeromonas hydrophila were reported for the first time in the bighead catfish, Clarias macrocephalus Günther, 1864 which is an important parental species for the production of the commercially important hybrid C. macrocephalus × C. gariepinus. The analyses were performed on 736 data records obtained from 74 full-sib families (31 half-sib families) produced by factorial mating design. The results showed that the heritability of survival rate after disease (Aeromonas hydrophila) challenge (intraperitoneal injection with 0.1 ml containing 1 × 106 CFU/ml of A. hydrophila) was low to moderate (0.05 ± 0.02–0.27 ± 0.15). The immune traits (bactericidal activity-BA, lysozyme activity-LA, and alternative complement activity-ACH50) had low to moderate heritability (h2BA = 0.05 ± 0.02; h2LA = 0.16 ± 0.04; h2ACH50 = 0.31 ± 0.06) while heritability of hematocrit (Hct) was also low (h2Hct = 0.17 ± 0.04). The results suggested the possibility to improve resistance to A. hydrophila by selection, while the possibility to use immunity traits as indirect selection criteria for disease resistance is still unclear.

Molecular characterization of manganese superoxide dismutase (MnSOD) from sterlet Acipenser ruthenus and its responses to Aeromonas hydrophila challenge and hypoxia stress

A novel gene encoding the mitochondrial manganese superoxide dismutase from sterlet Acipenser ruthenus (Ar-MnSOD) was cloned. The full-length cDNA of MnSOD was of 1040 bp with a 672 bp open reading frame encoding 224 amino acids and the deduced amino acid sequence was located in mitochondria. Sequence comparison analysis showed that Ar-MnSOD was highly similar to MnSODs of invertebrates and vertebrates, especially those of freshwater Cyprinidae fishes and mammals. Phylogenetic analysis revealed that Ar-MnSOD was distant from MnSODs of other fishes and belonged to the family of mitochondrial MnSODs (mMnSOD). Consistently, Ar-MnSOD was located in mitochondria. The 3D structure of Ar-MnSOD was predicted and the overall structure was similar to that of MnSODs of humans and the bay scallop Argopecten irradians. In addition, mRNA of Ar-MnSOD was detected to extensively express in all tissues, with the highest level in brain and liver. Spleen and head kidney inoculation of Aeromonas hydrophila led to a significant up-regulation of Ar-MnSOD transcript levels. Also, hypoxia induced a transient increase in transcription of Ar-MnSOD in the gills, but not in the heart and brain, suggesting metabolic depression in these vital organs. The results also implied the anti-hypoxia properties of Ar-MnSOD in the related tissues and proved that Ar-MnSOD was involved in the stress response and (anti) oxidative processes triggered by hypoxia. The results indicated that Ar-MnSOD is induced upon A. hydrophila infection and hypoxia, consistent with its role in host immune and stress-induced anti-oxidative responses.

Efficacy of the dehydrated lemon peels on the immunity, enzymatic antioxidant capacity and growth of Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus)

This work was performed to investigate the effect of the dietary addition of lemon, Citrus limon (L.) Burm. peels on the growth performance, antioxidant activity, immune status, and disease resistance against Aeromonas hydrophila in two common freshwater fish species (Nile tilapia, Oreochromis niloticus and African catfish, Clarias gariepinus). The fishes were randomly distributed at 10 fish per 60 L aquariums, and fed on three different diets (a control basal diet and two supplemented diets with 1% and 2% dried C. limon peels powder) for 45 days. The growth performance was insignificantly decreased in Nile tilapia fed on 2% followed by 1% C. limon peels supplemented diets and in African catfish fed on 2% C. limon peels when compared with the control fishes. Serum glucose was decreased with increasing levels of C. limon peels (2% followed by 1%) in Nile tilapia diets, whereas no significant effects were observed in African catfish. The cortisol levels remained unchanged in both fish species. However, significant elevation (p < .05) in the level of catalase (CAT) enzyme was seen in both fish species fed on C. limon peels (1% and 2%). Superoxide dismutase (SOD) was significantly increased in African catfish fed on C. limon peels (1% and 2%), whereas no significant change was noted in Nile tilapia. The reduced glutathione (GSH) content was significantly increased in both fish species fed on 2% C. limon peels; whereas no significant change was detected when they fed on 1% C. limon peels. The C. limon peels supplemented diets enhanced the serum lysozymes activity, myeloperoxidase (MPO) level and the phagocytic activity in both fish species. Moreover, nitric oxide level was significantly increased in Nile tilapia fed on 1% and 2% C. limon peels, while no significant change was observed in African catfish. The relative percentage survival (RPS), post A. hydrophila challenge, was found to be boosted in both fish species fed on C. limon peels (1% and 2%), when compared with the control fishes. The results elucidated that the inclusion of C. limon peels in Nile tilapia and African catfish diets could improve the enzymatic antioxidant capacity and immune response, but no significant effect on growth performance was observed. Thus, C. limon peels as a by-product could be successfully used as a cheap feed additive in aquaculture to enhance disease resistance.

Thymosin participates in antimicrobial immunity in zebrafish

Thymosin hormones, which were shown to be involved in immune system development and differentiation in previous studies, have antimicrobial functions in different animals. Zebrafish are a useful model for immunology research. Although thymosin has been reported to be involved in the embryonic development of zebrafish, it is necessary to uncover the antimicrobial function of thymosin in zebrafish. In this study, we expressed thymosin β (Tβ) in zebrafish in vitro and studied its antimicrobial function. The Tβ protein consists of 45 amino acids and is conserved among its family members, especially the actin-binding motif (LKKTET). Tβ was expressed in all tested tissues and was highly expressed in the brain, liver and hindgut. After Aeromonas hydrophila challenge, the Tβ transcript level increased in the skin, liver, kidney, spleen, thymus, foregut, gills and midgut. Purified recombinant thymosin β (rTβ) protein was used to study the antimicrobial mechanism. rTβ could inhibit the growth of Staphylococcus aureus, Aeromonas hydrophila, Vibrio anguillarum, Pseudomonas aeruginosa and Klebsiella pneumoniae. rTβ also binds to and agglutinates certain bacteria. Further study showed that rTβ could combine with the polysaccharides from gram-negative and gram-positive bacterial walls. All results suggested that the Tβ of zebrafish plays a significant role in innate antibacterial immune responses.

Effects of Housefly Maggot Meal and Earthworms on Growth and Immunity of the Asian Swamp Eel Monopterus albus (Zuiew)

Fly larvae (maggot meal) and earthworms are promising potential replacements for increasingly expensive and unsustainable fishmeal. Apart from being a good protein source, fly larvae also contain substances with immunostimulant capacity, however the impact of their inclusion in feed on the immune system remains only partially understood. To address this, we studied the effects of four diets with varying levels of earthworm (40-70%) and maggot meal (0-30%) on growth and immunocompetence of juvenile Asian swamp eels Monopterus albus (fish n = 480, duration = 40 days). Maggot meal inclusion resulted in a significantly (P&lt;0.05) higher total protein, glutamic pyruvic transaminase activity, anti-oxidizing enzyme and lysozyme activity, and significantly lower triglycerides, in the blood serum. Bacterial (Aeromonas hydrophila) challenge test results showed that adding maggot meal to the feed improved survival rate and immunocompetence at all three tested inclusion ratios. The strongest positive impact on most studied parameters (including 50% higher survival and 21.4% higher immunocompetence) was observed in the 20% inclusion group. As positive effects were slightly lower at the 30% level, the optimal combination of the two ingredients was 20% maggot meal and 50% earthworms

Feeding-regimen of β-glucan to enhance innate immunity and disease resistance of Nile tilapia, Oreochromis niloticus Linn., against Aeromonas hydrophila and Flavobacterium columnare

β-glucan is one of the most potent immunostimulants enhancing innate immune activity, disease resistance and growth performance of many aquatic organisms. Nevertheless, there are few studies on feeding regimens of β-glucan that correlate to immune response and disease resistance and are important considerations for practical β-glucan utilization. Thus, the effect of β-glucan and feeding duration on innate immunity and disease resistance was investigated to establish an optimal feeding regimen of β-glucan for Nile tilapia (Oreochromis niloticus Linn.). A variety of β-glucan feeding regimens were evaluated, including: i) feeding for 2 weeks, ii) feeding for 4 weeks, and iii) feeding every-other-week, with the objective of establishing the optimal feeding regimen that enhanced innate immunity and disease resistance. Innate immunity parameters were determined every week for eight weeks. Alternative complement activity of all β-glucan groups was significantly (P < 0.05) increased at the end of the first week, and then fluctuated but was not significantly (P > 0.05) different to the control until the end of the trial. Increased lysozyme activity was only detected at the end of the second week in all β-glucan-treated groups, and then decreased to the control level during most of the sampling periods. Phagocytosis percentage was increased and prolonged by β-glucan feeding, while the phagocytic index was not. Apart from innate immunity, β-glucan-fed fish demonstrated enhanced disease resistance against Aeromonas hydrophila and Flavobacterium columnare challenge at only the end of the fourth week of the trial. The growth performance of β-glucan-fed fish was not significantly (P > 0.05) different among the experimental groups and control. Taken together, the result indicated that all β-glucan-feeding regimens resulted in quite similar outcomes with respect to innate immunity stimulation, disease resistance and growth performance. This novel result suggests that an every-other-week regimen is the optimal choice for Nile tilapia cultivation as an economic cost saving benefit. This is the first study to determine the optimal feeding-regimen of β-glucan to enhance innate immunity and increase resistance to infection by pathogenic bacteria in Nile tilapia.

The comparisons in protective mechanisms and efficiencies among dietary α-lipoic acid, β-glucan and l-carnitine on Nile tilapia infected by Aeromonas hydrophila

Dietary α-lipoic acid (LA), β-glucan (Gluc) and l-carnitine (L-Ca) are commonly used additives to promote fish growth and stress resistance in aquaculture production. However their mechanisms and efficiencies in helping fish to resist diseases have not been compared before. In this study, we fed Nile tilapia (Oreochromis niloticus) with diets containing appropriate doses of LA, Gluc and L-Ca for five weeks and further intraperitoneally injected the fish with Aeromonas hydrophila. After dietary treatment, none of the additives affected the fish growth, but dietary Gluc and L-Ca reduced protein and lipid body contents in fish, respectively. After A. hydrophila challenge, all fish treated with the three dietary additives showed higher survival rate, but those fed on dietary L-Ca had lower survival than those fed on LA and Gluc diets, indicating high protection efficiency of LA and Gluc. The protective mechanisms of the three feed additives were quite different under A. hydrophila infection. Dietary LA induced higher total antioxidant capacity and higher mRNA expression of anti-oxidative genes than other additives in liver and also activated partly the immune function in serum and spleen. Gluc largely increased the immune function by activating the immunity enzymes in serum, inducing inflammation in liver and increasing the expression of immune genes in spleen and head kidney. Gluc also increased partly the antioxidant capacity in serum and liver and lipid catabolism in liver. L-Ca largely increased lipid catabolism in liver while it increased partly the antioxidant capacities in serum and liver. Taken together, these results indicate that, dietary LA, Gluc and L-Ca have various protective mechanisms and differ in their efficiencies on resisting A. hydrophila infection in Nile tilapia.

Effects of inosine 5′-monophosphate supplementation in high fishmeal and high soybean diets on growth, immune-related gene expression in gibel carp (Carassius auratus gibelio var. CAS Ⅲ), and its challenge against Aeromonas hydrophila infection

The present study was conducted to evaluate dietary inosine 5′-monophosphate (5′-IMP) on growth, immune genes expression and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS Ⅲ) (initial body weight: 7.48 g). Six diets were formulated containing exogenous 5′-IMP at three gradient levels (0, 0.1% and 0.2%) in the high dietary fishmeal group (15% fishmeal: D1, D2, D3) and in the high dietary soybean meal group (33% soybean meal: D4, D5, D6). Each diet was randomly allotted to triplicate tanks in a recirculating system. After the feeding trial, fish were exposed to Aeromonas hydrophila challenge. Hematological and immunological responses were analyzed before and after challenge. The results indicated that feeding rate in all 5′-IMP supplemented treatments (D2, D3, D5 and D6) and daily growth coefficient in D5 and D6 were reduced compared with those of respective control treatments (D1 and D4) without 5′-IMP addition (P < 0.05). The cumulative survival rates were numerically improved by dietary 5′-IMP supplementation (P > 0.05). Compared with the respective control treatment, in the high fishmeal group, plasma SOD and MPO were significantly elevated in D3 at the end of feeding trial (P < 0.05), plasma SOD and lysozyme were significantly increased in D3 after bacterial challenge (P < 0.05); in high soybean meal group, plasma lysozyme activity was significantly elevated in D5 post bacterial challenge (P < 0.05). Most of the expression of immune related genes (intelectin, major histocompatibility complex class II β (MHC II β), Complement 3 (C3), Complement component C7-1 (ccC7), lysozyme C, Interleukin 1β (IL-1β), Tumor necrosis factor α1 (TNF-α1), Transforming growth factor-beta (TGF-β) and Interleukin 8 (IL-8)) in spleen, kidney and liver of the fish were significantly affected by supplementation of 5′-IMP at the end of feeding trial and post bacterial challenge. Additionally, adding 5′-IMP in high soybean meal diets exerted further effects of promoting immunity than counterparts in high fishmeal diets. Considering enhanced disease resistance, the immunopotentiation of 5′-IMP was manifested when the addition level was 0.1% in high soybean meal diets and 0.2% in high fishmeal diets.