Liangkun Pan

Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih)

Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were alpha-helix and random coil. Fluorescent real-time quantitative RT-PCR was used to study the effects of heat shock (34 degrees C), crowding stress (100g L(-1)) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.

Effects of anthraquinone extract from Rheum officinale Bail on the growth performance and physiological responses of Macrobrachium rosenbergii under high temperature stress.

In order to study the effects of anthraquinone extract from Rheum officinale Bail on Macrobrachium rosenbergii under high temperature stress, freshwater prawns were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 0.05%, 0.1%, 0.2%, and 0.4% anthraquinone extracts for 10 weeks, respectively. Then, freshwater prawns were exposed to high temperature stress at 35 degrees C for 48h. The growth, changes in haemolymph total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lysozyme, nitrogen monoxide (NO) and hepatic catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) were investigated. The results showed that compared the control group, the specific growth rates, feed conversion efficiency, haemolymph ALP and lysozyme activities, total protein contents, hepatic CAT and SOD activities increased while haemolymph AST, ALT and hepatic MDA contents decreased in treatment groups before the stress, but their levels did not correlate with the doses of anthraquinone extracts. The specific growth rate (SGR), feed conversion efficiency and haemolymph lysozyme activity significantly increased but haemolymph AST activity decreased in 0.1% dose group; whereas haemolymph ALP activity and feed conversion efficiency increased but ALT activity and hepatic MDA contents significantly decreased in 0.2% dose group before the stress compared with the control. After high temperature stress, 0.1-0.2% anthraquinone extract also could improve the haemolymph total proteins, lysozyme and ALP activities, hepatic catalase, and superoxide dismutase, and reduce haemolymph ALT and AST activities, hepatic malondialdehyde contents. The cumulative mortality in the control was about 100% at 48h after high temperature stress while the cumulative mortality in the treatment groups supplemented with 0.1-0.2% anthraquinone extract were about 48-65%. The artificial infection with Vibrio anguillarum also showed the cumulative mortality in the control was about 100% while the cumulative mortality in the treatment groups supplemented with 0.1-0.2% anthraquinone extracts were about 57-80%. The present study suggested that ingestion of a basal diet supplemented with 0.1-0.20% anthraquinone extracts could prevent high temperature stress and promote the growth of prawns.

Effects of mannan oligosaccharide on the physiological responses, HSP70 gene expression and disease resistance of Allogynogenetic crucian carp (Carassius auratus gibelio) under Aeromonas hydrophila infection

We evaluated the effect of dietary supplementation with mannan oligosaccharide (MOS) on the resistance to Aeromonas hydrophi1a infection in Allogynogenetic crucian carp. The fish were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 60, 120, 240, 480 mg/kg MOS for 10 weeks, respectively. We then challenged the fish with A. hydrophi1a and recorded the mortality and the changes in serum cortisol, T3, T4, lysozyme, alkaline phosphatase (ALP), globin and hepatic total anti-oxidative capacity, superoxide dismutase (SOD), malondialdehyde (MDA) and the relative expression of heat shock protein 70 (HSP70) mRNA for a period of 7 d. Supplementation with 240 mg/kg MOS significantly increased serum ALP activity before infection, 1d and 2d after infection, serum globin concentration prior to infection, 1d and 7d after infection, serum lysozyme activity at 2d after infection, T3 concentration at 2d after infection, hepatic total anti-oxidative capacity prior to infection, hepatic SOD activity at 7d after infection and reduced serum cortisol concentration at 2d after infection, hepatic malondialdehyde content at 1d and 2d after infection. Supplementation with 480 mg/kg MOS significantly increased serum ALP activity before infection, 1d and 2d after infection, T3 content 1d after infection, T4 content prior infection and 7d after infection, serum globin concentration prior to infection, 1d and 7d after infection, serum lysozyme activity prior infection and 1d after infection, serum total anti-oxidative capacity prior to infection and 7d after infection, hepatic SOD activity at 7d after infection and the relative level of hepatic HSP70 mRNA at 2d and 7d after infection, had decreased levels of serum cortisol concentration before the infection, at 2d after infection, T4 concentration at 1d and 2d after infection, hepatic malondialdehyde content at 1d and 2d after infection. Mortality was significantly lower in the group of 240 and 480 mg/kg MOS than the control. Our results suggest that ingestion of a basal diet supplemented with 240-480 mg/kg MOS can enhance resistance against pathogenic infections in Allogynogenetic crucian carp.

A deficiency or an excess of dietary threonine level affects weight gain, enzyme activity, immune response and immune-related gene expression in juvenile blunt snout bream (Megalobrama amblycephala).

A feeding trial was conducted to investigate the impacts of deficient and excess dietary threonine levels on weight gain, plasma enzymes activities, immune responses and expressions of immune-related genes in the intestine of juvenile blunt snout bream. Triplicate groups of fish (initial weight 3.01 ± 0.01 g, 30 fish per tank) were fed with deficient (0.58%), optimum (1.58%) and excess (2.58%) threonine level diets to near satiation four times a day for 9 weeks. A mixture of l-amino acids was supplemented to simulate the whole body amino acid pattern of blunt snout bream, except for threonine. The results showed that both deficiency and excess threonine level diets significantly (P < 0.05) reduced the weight gain of blunt snout bream. Excess dietary threonine level triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (P < 0.05); whereas superoxide dismutase (SOD) activity was not significantly influenced by imbalanced-dietary threonine level (P > 0.05). Plasma complement component 3 (C3) and component 4 (C4) concentrations were significantly depressed by the deficiency of dietary threonine (P < 0.05). Dietary threonine regulated the target of rapamycin (TOR), eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), tumour necrosis factor alpha (TNF-α) and copper-zinc superoxide dismutase (Cu/Zn-SOD) gene expressions in the intestine of blunt snout bream, which may go further to explain the adverse effects of a deficient and/or an excess dietary threonine level on growth, immunity and health of fish. Furthermore, the present study also suggests that an optimum dietary threonine could play an important role in improving growth, enhancing immune function and maintaining health of fish.

Effects of emodin and vitamin E on the growth and crowding stress of Wuchang bream (Megalobrama amblycephala).

This study aimed to investigate the effects of dietary emodin, high-dose vitamin E and their combination on the growth of Megalobrama amblycephala and its resistance to acute crowding stress. The fish were randomly divided into four groups: a control group fed with basal diet, and three treatment groups fed with basal diet supplemented with 60 mg/kg emodin (the emodin group), 500 mg/kg vitamin E (the vit E group), and 60 mg/kg emodin together with 500 mg/kg vitamin E (the combination group). After 60 days, the fish were exposed to acute crowding stress for 24 h. The results showed that the weight gain of the vit E group, specific growth rate of the vit E group, total serum protein concentration (TP) of the vit E group, serum lysozyme activity of the emodin group, serum superoxide dismutase (SOD) activity of the emodin group, hepatic heat shock protein 70 (HSP70) levels of the vit E group and the emodin group, and serum alanine aminotransferase (ALT) activity of the combination group significantly increased while the weight gain and specific growth rate of the combination group significantly decreased compared with the control group before stress. After crowding stress, the vit E group had improved serum TP 12 h post-stress, hepatic SOD activity 24 h post-stress, and hepatic HSP70 mRNA levels 12 and 24 h post-stress while the emodin group had enhanced serum SOD activity 12 and 24 h post-stress and hepatic HSP70 mRNA levels 12 and 24 h post-stress, as compared with the control. However, the serum cortisol content of the three treatment groups 12 and 24 h post-stress, ALT activity in the vit E group and emodin group 24 h post-stress, and serum alkaline phosphatase and liver catalase activity in the combination group 24 h post-stress were lower than those in the control group. The cumulative mortality was lower in the emodin, vit E, and combination group after Aeromonas hydrophila infection compared with the control group. Therefore, dietary supplementation with 60 mg/kg emodin or 500 mg/kg vitamin E can improve HSP70 mRNA levels and antioxidant capabilities, resistance to crowding stress, and growth in M. amblycephala. However, the combination of emodin and vit E does not have a synergistic effect in M. amblycephala.

Mass mortality of pond-reared Carassius gibelio caused by Myxobolus ampullicapsulatus in China.

From June to August 2009, allogynogenetic silver crucian carp Carassius gibelio (Bloch) pond-cultured at the Nanquan Experimental Station, China, were found to be heavily infected with myxosporeans, which caused mortalities ranging from 33% (13/40) to 90% (36/40) in the cages. The pharynxes of infected fish were swollen, nodular, and severely damaged. Based on morphological characters and 18S small subunit ribosomal DNA sequence similarity, the myxosporean was identified as Myxobolus ampullicapsulatus. This is the first report of M. ampullicapsulatus causing mass mortality of pond-reared C. gibelio.

Threonine affects digestion capacity and hepatopancreatic gene expression of juvenile blunt snout bream ( Megalobrama amblycephala )

The present study conducted a 9-week feeding trial to investigate the effects of threonine (Thr) on the digestion capacity and hepatopancreas gene expression of juvenile blunt snout bream (Megalobrama amblycephala). For this purpose, three tanks (300 litres/tank) were randomly arranged and assigned to each experimental diet. Juvenile fish were fed with diets containing graded Thr levels (0·58, 1·08, 1·58, 2·08 or 2·58 % of the diet) to apparent satiation four times daily. At the end of the feeding trial, the results indicated that hepatopancreas weight, hepatosomatic index, hepatopancreatic protein content, intestinal weight, intestosomatic index and intestinal protein content increased with increasing dietary Thr levels up to 1·58 % and thereafter decreased (P< 0·05). The activities of chymotrypsin, trypsin, amylase and lipase elevated as dietary Thr levels increased up to 1·58 % (P< 0·05), while these activities decreased in most cases after 1·58 % dietary Thr except for chymotrypsin and trypsin in the hepatopancreas (plateau 1·58-2·08 % Thr). The relative gene expression levels of chymotrypsin, trypsin, amylase, lipase, target of rapamycin and insulin-like growth factor-I were up-regulated, and the highest values were observed with 1·58 % dietary Thr or 1·58 and 2·08 % dietary Thr, whereas the relative gene expression levels of eukaryotic translation initiation factor 4E-binding protein 2 gradually decreased (P< 0·10) as dietary Thr levels increased up to 1·58 % and thereafter significantly increased (P< 0·05), which could explain that about 1·58 % dietary Thr could improve the growth and development of digestive organs and activities of digestive enzymes of juvenile blunt snout bream.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

&NA; Nitrite (NO2−) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ± 0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and 12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH‐Px), glutathione S‐transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up‐regulated the mRNA levels of glutathione peroxidase (GSH‐Px), glutathione S‐transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre‐stress levels. The results suggested that the glutathione system played important roles in nitrite‐induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH‐Px, GST, GR and up‐regulating the expression level of GSH‐Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage. Graphical abstract Figure. No caption available. HighlightsNitrite exposure induced oxidative stress and tissue damaged on the liver of Aristichthys nobilis.Upregulation of GSH‐Px, GST, GR mRNA expressions and enzyme activities maintained the dynamic balance of GSH/GSSG system.The GSH/GSSG system fighted against oxidative injury caused by nitrite stress.

Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds

We surveyed the actinosporean stages of fish myxosporeans at fish farms in Jiangsu Province, China, from 2011 to 2014. During the surveys, we identified 7 actinosporean types from 4 collective groups: echinactinomyxon (1 type), triactinomyxon (1 type), aurantiactinomyxon (1 type), and neoactinomyxum (4 types), released by the oligochaete Branchiura sowerbyi. The morphological characteristics and DNA sequences of these types are described here. Based on 18S rDNA sequence analysis, the actinosporean of echinactinomyxon type CZ with 4 branches at the end of the caudal processes was identified as Myxobolus wulii, and the neoactinomyxum type JD was identified as Thelohanellus wangi Yuan, Xi, Wang, Xie, Zhang, 2015 (JX458816), a recently nominated species from the gills of allogynogenetic gibel carp Carassius auratus gibelio. In addition, actinosporeans of aurantiactinomyxon type JD, neoactinomyxum type CZ-1, neoactinomyxum type CZ-2, and neoactinomyxum type CZ-3 showed high genetic similarity to T. wuhanensis (96.3-96.5%), T. nikolskii (98.0-99.1%), T. wuhanensis (97.8-98.9%), and T. hovorkai (98.7-98.9%), respectively. Phylogenetic analyses showed that these actinosporeans were robustly clustered in the Thelohanellus spp. clade.

Effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream

ABSTRACT The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK‐NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T‐NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%–2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%–2.70% dietary arginine levels in enzyme‐linked immune sorbent assay. At dietary arginine levels of 1.22%–2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL‐8) and tumour necrosis factor‐&agr; (TNF‐&agr;) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro‐inflammatory factor including IL‐8 and TNF‐&agr; and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T‐SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1&bgr; (IL‐1&bgr;) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn‐SOD), manganese superoxide dismutase (Mn‐SOD) and IL‐1&bgr; were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%–1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T‐NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%–2.70% dietary arginine levels in enzyme‐linked immune sorbent assay. At dietary arginine levels of 1.96%–2.31%, T‐SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%–2.70% dietary arginine levels. At dietary arginine levels of 1.22%–2.31%, the plasma TNF‐&agr; and IL‐8 contents were significantly decreased. Significantly lower plasma IL‐1&bgr; contents were observed in fish fed 1.62%–1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro‐inflammatory genes including Cu/Zn‐SOD, Mn‐SOD, GPx, IL‐8, TNF‐&agr; and IL‐1&bgr;. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%–1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%–2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro‐inflammatory genes including IL‐8, TNF‐&agr; and IL‐1&bgr; and plasma MDA, IL‐8, TNF‐&agr; and IL‐1&bgr; contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine‐AMPK‐NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream. HIGHLIGHTSDietary arginine supplementation could increase NO production in M. amblycephala.Optimal dietary arginine level (1.62%–1.96%) could improve antioxidant capacity by AMPK‐NO signaling pathway.Optimal dietary arginine level could improve immune response and weaken tissues inflammatory.High arginine level (2.70%) resulted in excessive NO production, which has a negative effect on M. amblycephala.