Differential effects of oxytetracycline on detoxification and antioxidant defense in the hepatopancreas and intestine of Chinese mitten crab under cadmium stress.

This study aims to evaluate the effects of oxytetracycline (OTC) on detoxification and oxidative defense in the hepatopancreas and intestine of Chinese mitten crab (Eriocheir sinensis) under cadmium (Cd) stress. The crab was exposed to 0.6 µM Cd, 0.6 µM OTC, and 0.6 µM Cd plus 0.6 µM OTC for 42 days. Our results showed that in the intestine, OTC alone enhanced protein carboxylation (PC) and malondialdehyde (MDA) contents, which was associated with the increased OTC accumulation. Compared to Cd alone, Cd plus OTC increased Cd and OTC contents, and reduced detoxification (i.e., glutathione (GSH) content, gene expressions of cytochrome P450 (CYP) isoforms, 7-ethoxyresorufin O-deethylase (EROD) activity, mRNA levels and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST)), and antioxidant defense (i.e., gene expressions and activities of catalase (CAT) and superoxide dismutase (SOD)) in the intestine, leading to the increased in PC and MDA contents, suggesting that OTC had a synergistic effect on Cd-induced oxidative damage. In the hepatopancreas, although OTC alone increased OTC accumulation, it did not affect PC and MDA contents. Compared to Cd alone, Cd plus OTC reduced MDA content, which was closely related to the improvement of detoxification (i.e., GSH content, mRNA levels of CYP isoforms, EROD activity, gene expressions and activities of GPx, GR and GST), and antioxidant defense (gene expressions and activities of CAT and SOD, metallothionein content). Aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2) transcriptional expressions were positively correlated with most detoxification- and antioxidant-related gene expressions, respectively, indicating that AhR and Nrf2 were involved in the regulation of these gene expressions. Our results unambiguously demonstrated that OTC had tissue-specific effects on Cd-induced toxicological effect in E. sinensis, which contributed to accurately evaluating Cd toxicity modulated by TCs in crab.

Effects of Broad Bean Diet on the Growth Performance, Muscle Characteristics, Antioxidant Capacity, and Intestinal Health of Nile Tilapia (Oreochromis niloticus).

Four crisping diets were designed to conduct a feeding experiment to investigate the use of broad bean in the formulated feed of Nile tilapia and optimize its use. The growth performance, muscle characteristics, antioxidant capacity, and intestinal health of Nile tilapia with an initial body weight of 617.32 ± 1.64 g were evaluated after being fed with different diets for 90 days. The results showed no adverse effect on the growth performance of Nile tilapia fed with broad bean diets. Contrastingly, some improvements were found in WGR and SGR, but a lower FCR was obtained. The supplementation of broad beans weakened the antioxidant capacity of fish but did not influence liver health and the immune system. Increasing the amount of broad bean addition can increase muscle quality values, and an embrittling functional package being added to the diet can also improve muscle hardness, adhesiveness, and chewiness of Nile tilapia muscle. In addition, the crisping functional package can be applied to tilapia crisping formulated feed, which helps to improve the integrity of the intestinal tissue structure and optimize the intestinal microbiota of Nile tilapia. The final achievement of this study is to provide a theoretical reference for optimizing the breeding technology of crispy Nile tilapia and developing a specialized crisping diet for the species.

Different effects of continuous and pulsed Benzo[a]pyrene exposure on metabolism and antioxidant defense of large yellow croaker: Depend on exposure duration.

This study aims to compare differential effects of continuous and pulsed BaP exposures on metabolism and antioxidant defense in the liver of large yellow croaker. Fish were subjected to BaP for 4 days and 36 days in three exposure regimes with the same time-averaged concentration of BaP: 4 µg/L BaP continuously, 8 µg/L BaP for 24 h every other day or 16 µg/L BaP for 24 h every 4 days. Our results showed that compared to pulsed BaP exposures, continuous BaP exposure reduced BaP metabolism (CYP1A, CYP3A and AHR transcriptional expressions, GSH content, GSH/GSSG ratio, EROD and GST activities) and antioxidant defense (T-SOD activity) on day 4, resulting to the increases in MDA and PC contents, indicating that continuous BaP exposure induced more severe oxidative damage during the early stage of exposure. But continuous BaP exposure reduced MDA and PC contents by improving BaP metabolism and antioxidant defense during the late stage of exposure. CYP1B transcriptional expression and CAT activity were unsuitable biomarkers of both continuous and pulsed BaP exposures. In conclusion, our results demonstrated differential effects of continuous and pulsed exposures on BaP metabolism and antioxidant responses, which were depend on exposure duration.

A broad-host-range lytic phage vB_VhaS-R18L as a candidate against vibriosis.

Vibriosis is one of the most common bacterial diseases that cause high rates of mortality and considerable economic losses in aquaculture. Phage therapy has been considered as a promising alternative method to antibiotics in the biocontrol of infectious diseases. Genome sequencing and characterization of the phage candidates are prerequisites before field applications to ensure environmental safety. In this study, a lytic phage, named vB_VhaS-R18L (R18L), was isolated from the coastal seawater of Dongshan Island, China. The phage was characterized in terms of morphology, genetic content, infection kinetics, lytic profile, and virion stability. Transmission electronic microscopy indicated that R18L is siphovirus-like, comprising an icosahedral head (diameter 88.6 ± 2.2 nm) and a long noncontractile tail (225 × 11 nm). Genome analysis indicated R18L to be a double-stranded DNA virus with a genome size of 80,965 bp and a G + C content of 44.96%. No genes that encode known toxins or genes implicated in lysogeny control were found in R18L. A one-step growth experiment showed that R18L had a latent period of approximately 40 min and a burst size of 54 phage particles per infected cell. R18L showed lytic activity against a wide range of at least five Vibrio species (V. alginolyticus, V. cholerae, V. harveyi, V. parahemolyticus, and V. proteolyticus). R18L was relatively stable at pH 6-11 and at temperatures ranging from 4°C to 50°C. The broad lytic activity across Vibrio species and the stability in the environment make R18L a potential candidate for phage therapy in controlling vibriosis in aquaculture systems.

Different cold tolerances among three strains of large yellow croaker: related to antioxidant defense and energy metabolism.

The aim of this study was to compare low-temperature tolerances in different strains of large yellow croaker. Dai Qu (DQ), Min-Yue Dong (MY), and Quan Zhou (NZ) strains of large yellow croaker were subjected to cold stress (8.6 °C) for 12 h, 24 h, 48 h, and 96 h. Survival rate, histological observation, and antioxidant and energy metabolism indicators were determined. The results showed that compared with the DQ group and MY group, NZ group aggravated hepatic structure, enhanced ROS, lactate, and anaerobic metabolism (PK gene expression and activity), while inhibited ATP, GSH, antioxidant enzymes (mRNA levels and activities of SOD, GPx, and CAT), and aerobic metabolism enzymes (mRNA levels and activities of F-ATPase, SDH, and MDH), indicating the reduction of cold tolerance in the NZ group was closely correlated with the decrement of antioxidative capacity and energy metabolism efficiency. Nrf2 and AMPK gene expressions were correlated with antioxidant and energy metabolism mRNA levels, respectively, suggesting Nrf2 and AMPK might participate in the modulation of target genes during the cold-stress adaptation. In conclusion, low temperature tolerance of fish depended on the antioxidant defense and energy metabolism efficiency, which contributes to understanding the underlying mechanisms of cold adaptation in large yellow croaker.

Treatments of orange-spotted grouper (Epinephelus coioides) against Cryptocaryon irritans with •OH, ClO2 or HCHO: Survival, physiological and histological response.

Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.

Autochthonous Probiotics Alleviate the Adverse Effects of Dietary Histamine in Juvenile Grouper (Epinephelus coioides).

High dose (0.3%) of dietary histamine can cause adverse effects on growth performance, innate immunity, and gut health in juvenile grouper (Epinephelus coioides). In the present study, three autochthonous probiotics (Bacillus pumilus SE5, Psychrobacter sp. SE6, and Bacillus clausii DE5) were supplemented separately to diets containing 0.3% of histamine and their effects on growth performance, innate immunity, and gut health of grouper (E. coioides) were evaluated in a 56-day feeding trial. The results showed considerable increase in weight gain, specific growth rate, hepatosomatic index, and decreased feed conversion rate in groupers fed with probiotic-supplemented diets. Supplementation of autochthonous probiotics has improved antioxidant capacity and innate immunity of E. coioides by measuring correlative parameters, such as total antioxidant capacity, superoxide dismutase activity, malondialdehyde content, and so on. Additionally, dietary probiotics have significantly reduced the levels of serum interleukin-1ß (at days 28 and 56), fatty acid-binding protein 2, and intestinal trefoil factor (at day 28), and promoted intestinal integrity following remarkably increased muscle thickness and mucosal fold height at day 56, especially in grouper fed with B. pumilus SE5 containing diet (P < 0.05). On day 56, the gut microbial composition of E. coioides was positively shaped by autochthonous probiotics, the relative abundance of potentially pathogenic Photobacterium decreased while beneficial Lactobacillus increased in fish fed with probiotic strains, especially with B. pumilus SE5 and B. clausii DE5. These results suggest that among the three autochthonous probiotic strains tested, B. pumilus SE5 is showing better efficiency in alleviating the adverse effects of (high levels) dietary histamine by decreasing the expression of inflammatory markers and by improving the growth, innate immunity, and gut health of juvenile grouper E. coioides.

Hypoxic acclimation improves mitochondrial bioenergetic function in large yellow croaker Larimichthys crocea under Cu stress.

The purpose of this study was to investigate how pre-hypoxia exposure affected the mitochondrial structure and bioenergetic function of large yellow croaker in responding to Cu stress. Fish were acclimated to normoxia and 3.0 mg DO L-1 for 48 h, then subjected to 0 and 120 µg Cu L-1 for another 48 h. Hypoxic acclimation did not affect mitochondrial ultrastructure and reactive oxygen species (ROS), but reduced oxidative phosphorylation (OXPHOS) efficiency. Cu exposure impaired mitochondrial ultrastructure, increased ROS generation and inhibited OXPHOS efficiency. Compared with Cu exposure alone, hypoxic acclimation plus Cu exposure reduced ROS production and improved OXPHOS efficiency by enhancing mitochondrial respiratory control ratio, mitochondrial membrane potential, and activities and gene expressions of electron transport chain enzymes. In conclusion, hypoxic acclimation improved the mitochondrial energy metabolism of large yellow croaker under Cu stress, facilitating our understanding of the molecular mechanisms regarding adaptive responses of hypoxia-acclimated fish under Cu stress.

Molecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

Modulation of copper-induced antioxidant defense, Cu transport, and mitophagy by hypoxia in the large yellow croaker (Larimichthys crocea).

This study aimed to investigate the effects of hypoxia on Cu-induced antioxidant defense, Cu transport, and mitophagy in the liver of the large yellow croaker. Fish were exposed to hypoxia (3.0 mg L-1), Cu (120 µg L-1), and hypoxia (3.0 mg L-1) plus Cu (120 µg L-1) for 48 h. Hypoxia exposure increased antioxidant abilities to maintain cellular redox balance. Although Cu exposure alone improved antioxidant defense, Cu transport, and mitophagy, these stress responses could not completely neutralize Cu toxicity, as reflected by the elevated reactive oxygen species (ROS) and lipid peroxidation (LPO) and hepatic vacuoles. When compared with Cu stress alone, hypoxia increased Cu toxicity by inhibiting antioxidant defense, Cu transport, and mitophagy, leading to the increment of mortality, ROS, and LPO, and the deterioration of histological structure. The adverse effects of hypoxia on Cu-induced metal transport and mitophagy might be involved in metal-responsive element-binding transcription factor-1 (MTF-1) and Forkhead box O-3 (FoxO3) signaling pathways, respectively. Overall, hypoxia reduced antioxidant response, Cu transport, and mitophagy in fish exposed to Cu, which contributes to understanding the molecular mechanisms underlying negative effects of hypoxia on Cu toxicity in fish.

Pre-hypoxia exposure inhibited copper toxicity by improving energy metabolism, antioxidant defence and mitophagy in the liver of the large yellow croaker Larimichthys crocea.

This study investigated the effects of moderate hypoxia pre-exposure on energy metabolism, antioxidant defence and mitophagy in the liver of the large yellow croaker Larimichthys crocea exposed to Cu. Fish were pre-exposed to either normoxia or hypoxia (~3.0 mg L-1, 42% O2 saturation) for 48 h, and subsequently were subjected to either control (without Cu addition) or Cu (168 µg L-1) under normoxic conditions for another 48 h. Copper exposure under normoxia induced Cu toxicity that increased mortality, the production of reactive oxygen species (ROS) and malondialdehyde, and aberrant hepatic mitochondrial ultrastructure. Interestingly, hypoxia pre-exposure improved energy metabolism, antioxidant ability and mitophagy response, and reduced the Cu content to inhibit Cu toxicity, reflecting the enhanced survival rate and reduced oxidative damage. In these processes, hypoxia-inducible factor-1α (HIF-1α), transcription factors NFE2-related nuclear factor 2 (Nrf2), and forkhead box O-3 (FoxO3) mRNA levels were correlated with expression of genes related to energy metabolism, antioxidant defence and mitophagy, respectively, indicating HIF-1α, Nrf2, and FoxO3 are required for the induction of their respective target genes. Overall, moderate hypoxia pre-exposure was able to generate adaptive responses to mitigate Cu-induced toxicological effects, underlining a central role of hormesis.

Cu pre-exposure alters antioxidant defense and energy metabolism in large yellow croaker Larimichthys crocea in response to severe hypoxia.

The aim of the present study was to evaluate the effects of Cu pre-exposure on antioxidant defense and energy metabolism in the liver of the large yellow croaker exposed to severe hypoxia. Fish were pre-acclimated to 0 and 30 µg Cu L-1 for 96 h, and subsequently exposed to 7.0 and 1.5 mg DO L-1 for another 24 h. Hypoxic stress alone increased reactive oxygen species and hepatic vacuoles. When compared to hypoxic stress alone, hypoxic stress plus Cu pre-exposure increased mortality and ROS production, and worsened histological structure by inhibiting antioxidant defense and aerobic metabolism, and enhancing anaerobic metabolism, suggesting Cu pre-acclimation aggravated hypoxia-induced oxidative damage. NFE2-related nuclear factor 2 and hypoxia-inducible factor-1α might participate in the transcriptional regulation of genes related to antioxidant response and energy metabolism, respectively. In conclusion, Cu pre-acclimation had a synergistic effect on antioxidant response and energy metabolism in fish under severe hypoxia, which contributes to understanding the molecular mechanisms underlying negative effects of Cu pre-acclimation against hypoxic damage in fish.

Identification and bioactivity of a granulocyte colony-stimulating factor b homologue from large yellow croaker (Larimichthys crocea).

Granulocyte colony-stimulating factor (GCSF) is a pleiotropic cytokine that plays a key role in regulation of hematopoiesis, innate and adaptive immune responses in mammals. However, bioactivity of GCSF in teleost fish remains largely unknown. In this study, a GCSFb homologue from large yellow croaker (Larimichthys crocea) (LcGCSFb) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcGCSFb is 603 bp long and encoded a protein precursor of 200 amino acids (aa), with a 19-aa signal peptide and a 181-aa mature peptide. In healthy fish, the LcGCSFb was constitutively expressed in all examined tissues, with the highest levels in mucous tissues, such as gills, intestine, and stomach. Its transcripts in head kidney, spleen, gills, intestine and stomach were significantly induced by Vibrio alginolyticus challenge. LcGCSFb transcripts were also detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM), primary head kidney granulocytes (PKG) and head kidney cell line (LYCK), and markedly upregulated by inactivated V. alginolyticus. These data suggested that LcGCSFb may play a role in immune response against bacterial infection. In vivo administration of recombinant LcGCSFb protein (rLcGCSFb) significantly upregulated the expression levels of the inflammatory cytokines (IL-6 and TNFα), and transcription factor C/EBPß, which is required for proliferation of neutrophils. Furthermore, rLcGCSFb showed an ability to strengthen the phagocytosis of PKL in vitro. Taken together, LcGCSFb may be involved in antibacterial immunity via promoting the inflammatory response and the phagocytic activity of leukocytes. To our knowledge, this is the first report on immunoregulatory roles of GCSF in teleost.

Toxicological effects of waterborne Zn on the proximal and distal intestines of large yellow croaker Larimichthys crocea.

The aim of the present study was to compare the differences of Zn-induced antioxidant defense, immunotoxicity and Zn homeostasis between the proximal and distal intestines of the large yellow croaker Larimichthys crocea. Fish were exposed to Zn (2 and 10 mg L-1) for 96 h. In the proximal intestine, high-concentration Zn increased mortality and oxidative damage, but reactive oxygen species (ROS), lipid peroxidation (LPO), protein carbonylation (PC) levels were not affected by low-concentration Zn, indicating Zn-induced oxidative damage was concentration-dependent. Antioxidant defense and immunotoxicity in response to Zn exposure may be involved in ROS/ NFE2-related nuclear factor 2 (Nrf2) and ROS/nuclear transcription factor κB (NF-κB) signaling pathways. In the distal intestine, Zn exposures did not induce oxidative damage, which may result from the improvement of Zn transport, antioxidant and immune defenses. Nrf2 was positively correlated with antioxidant-related gene in the distal intestine, but no relationship was observed between Nrf2 and CAT gene expressions in the proximal intestine. In conclusion, Zn induced toxicological effects were intestinal-region-dependent, which provided some novel insights into Zn toxicology.

Essential element Cu and non-essential element Hg exposures have different toxicological effects in the liver of large yellow croaker.

The objective was to compare the different effects of essential element Cu and non-essential element Hg on antioxidant and inflammatory responses in the liver of large yellow croaker Larimichthys crocea. Fish were exposed to Cu stresses (72 and 288 µg L-1) and Hg stresses (14 and 56 µg L-1) for 96 h. High-dose Cu increased metallothioneins (MTs) levels and immune defenses in response to elevated reactive oxygen species (ROS), but low-dose Cu had no effect on ROS. High-dose Hg reduced antioxidant and inflammatory responses, which contributed to the increment of ROS. MTs may be a suitable biomarker to assess Cu contamination, but no relationship was observed between MTs levels and Hg content. Furthermore, NFE2-related nuclear factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) were positively related to their respective target genes in the Cu-exposed groups. In conclusion, Cu and Hg induced some differences in antioxidant and inflammatory responses, which providing some novel insights into toxicological effects of Cu and Hg stresses.

Differential effects of ß-glucan on oxidative stress, inflammation and copper transport in two intestinal regions of large yellow croaker Larimichthys crocea under acute copper stress.

The aim of the present study was to evaluate investigate the effects of ß-glucan on oxidative stress, inflammation and copper transport in two intestinal regions of large yellow croaker under acute copper stress. Fish were injected with ß-glucan at a dose of 0 or 5 mg kg-1 body weight on 6, 4 and 2 days before exposed to 0 and 368 µg Cu L-1 for 48 h. Biochemical indicators (MDA, Cu content, MTs protein levels, Cu/Zn-SOD, CAT and iNOS activities), gene expressions of oxidative stresses (Cu/Zn-SOD, CAT, Nrf2, MTs and MTF-1), inflammatory responses (NF-κB, iNOS, IL-1ß, IL-6 and TNF-α) and Cu transporters (ATP7A, ATP7B and CTR1) were determined. In the anterior intestine, ß-glucan increased MTs levels, activities of Cu/Zn-SOD, CAT and iNOS, mRNA levels of MTs, CAT, iNOS, ATP7A and ATP7B, and reduced Cu content and CTR1 gene expression to inhibite Cu-induced MDA. But ß-glucan had no effect on inflammatory gene expressions. In the mid intestine, ß-glucan increased activities of Cu/Zn-SOD and iNOS, mRNA levels of Cu/Zn-SOD, CAT and iNOS to maintain MDA content. However, unlike the anterior intestine, ß-glucan had no effect on Cu transporter gene expressions. Furthermore, transcription factors (Nrf2, NF-κB and MTF-1) paralleled with their target genes in the mid intestine, but no correlation was observed between NF-κB and IL-1ß and TNF-α gene expressions in the anterior intestine. In conclusion, our results unambiguously showed that ß-glucan induced oxidative stress, inflammation and copper transport were varied between the anterior and mid intestines of fish under Cu stress.

Molecular cloning and bioactivity of an IL-2 homologue in large yellow croaker (Larimichthys crocea).

Interleukin-2 (IL-2), an important immunomodulatory cytokine, plays a crucial role in promoting the proliferation, activation and differentiation of T cells. Here, the cDNA of an IL-2 homologue (LcIL-2) in large yellow croaker (Larimichthys crocea) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcIL-2 gene is 426 bp long and encoded a precursor protein of 141 amino acids (aa), with a 20-aa signal peptide and a 121-aa mature peptide containing two putative N-glycosylation sites at Asn77 and Asn101. The LcIL-2 is preferentially expressed in lymphocytes-rich tissues, such as spleen and blood, and is increased in head kidney and spleen upon inactivated trivalent bacterial vaccine or poly(I:C) stimulation. LcIL-2 expression could also be detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM) and primary head kidney granulocytes (PKG), with the highest level in PKL. In addition, the expression level of LcIL-2 in PKL was slightly induced by LPS or poly(I:C), while markedly induced by PHA or Con-A. The recombinant LcIL-2 protein produced in Pichia pastoris could increase the expression of genes involved in Th1 (IL-2, IFN-γ and T-bet) and Th2 (IL-4/13A, IL-4/13B and GATA3) development and differentiation, and of the IL-2 downstream transcription factor STAT5B gene, but inhibit the expression of genes related to Th17 (IL-17A/F2 and IL-17A/F3) development and differentiation. Taken together, our results indicated that LcIL-2 possesses similar structural and functional characteristics to other vertebrate IL-2s, and may play a role in T cell development and differentiation.

Abrupt salinity stress induces oxidative stress via the Nrf2-Keap1 signaling pathway in large yellow croaker Pseudosciaena crocea.

The aim of the present study was to evaluate the effects of abrupt salinity stress (12, 26 (control), and 40) on lipid peroxidation, activities and mRNA levels of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecules at different times (6, 12, 24, and 48 h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced at 6 h and increased at 12 h before returning to control levels in the hypo-salinity group. Similarly, lipid peroxidation was significantly decreased at 6 h followed by a sharp increase towards the end of the exposure in the hyper-salinity group. Negative relationships between lipid peroxidation and antioxidant enzyme activities and positive relationships between activities and gene expression of antioxidant enzymes were observed, suggesting that the changes at molecular levels and enzyme activity levels may provide protective roles against damage from salinity stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for regulating these genes. Furthermore, there was a positive relationship between the mRNA levels of Nrf2 and Keap1, indicating that Keap1 plays an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of salinity stress on antioxidant responses in large yellow croaker through the Keap1-Nrf2 pathway.

Molecular characterization and evolution analysis of five interleukin-17 receptor genes in large yellow croaker Larimichthys crocea.

Interleukin-17s (IL-17s) play critical roles in inflammatory response and host defense against extracellular pathogens. IL-17s induce the immune response signaling through the specific IL-17 receptors (IL-17Rs) that consist of five members (IL-17RA to E). In the present work, we have identified the five IL-17R orthologs (LycIL-17Rs) from large yellow croaker Larimichthys crocea. The deduced protein of each LycIL-17R exhibits a typical IL-17R domain architecture, including a signal peptide, the extracellular FNIII domain (IL-17RA/RB/RD) or IL-17_R_N domain (IL-17RC/RE), a transmembrane domain, and a SEFIR domain in cytoplasmic region. In particular, the extracellular regions of teleost IL-17RB are much shorter than those in mammals and lack an FNIII domain (FN2). Phylogenetic tree shows that IL-17Rs are classified into two main groups: IL-17RA/RB/RD group and IL-17RC/RE group, which is distinct from previous proposal that grouped IL-17RB into IL-17RC/RE. The surrounding genes of IL-17Rs are conservatively aligned in genomes between teleosts and mammals. The five LycIL-17Rs were constitutively expressed in all tissues examined, but with different expression patterns. Aeromonas hydrophila infection significantly upregulated LycIL-17RA, RC, RD and RE in both mucosal tissue (gills) and systemic immune tissues (head kidney and spleen), while the increase of LycIL-17RB expression could be detected in gills, indicating that LycIL-17Rs may be involved in host defense against bacterial infection. Thus, these results suggest that teleost IL-17Rs may function in mediating immune response as their mammalian orthologs. To our knowledge, this is the first report of molecular characterization of the five IL-17Rs (IL-17RA/RB/RD and IL-17RC/RE) in teleost fish.

Effects of ß-glucan on ROS production and energy metabolism in yellow croaker (Pseudosciaena crocea) under acute hypoxic stress.

The aim of the present study was to evaluate the effect of ß-glucan on acute hypoxia-induced oxidative stress and the changes in energy metabolism by determining ROS production, activities and mRNA levels of energy metabolism enzyme (PK, F-ATPase, SDH and MDH), and in gene expression of HIF-1α in the liver of large yellow croaker. Fish were injected with ß-glucan at a dose of 0 or 5 mg kg(-1) body weight on 6, 4 and 2 days before exposed to 1.5 and 7.0 mg DO L(-1) for 48 h. The results showed that ß-glucan enhanced survival rate and reduced ROS during the lethal hypoxic stress, indicating that ß-glucan could ameliorate hypoxia-induced oxidative stress. Obtained results also showed that ß-glucan could up-regulate activities and mRNA levels of PK, demonstrating that ß-glucan increased anaerobic glycolysis capacity. Furthermore, a coordinated transcriptional regulation of energy metabolism enzyme genes was observed, suggesting that HIF-1α is required for regulating these genes. In conclusion, ß-glucan could alleviate cute hypoxia-induced oxidative stress in large yellow croker by enhancing anaerobic glycolysis capacity, emphasizing a central role of transcription factor HIF-1α in the process.