Effects Of High Temperature Stress And Anthraquinone Extract From Rheum Officinale Bail On The Physiological Response And Gene Expression Of Stress Heat Shock Protein Of Wuchang Bream(Megalobrama Amblycephala)

In intensive aquaculture, fish is constantly exposed to various stressors such as ambient temperature, stocking density, physicochemical parameters, transport, and confinement.The feasibility of disease and infection is facilitated by various stressor when pathogens find an adequate environment for reproduction. Fish are poikilotherms and the immune response against invading pathogens depends much more on the environmental temperature. Temperature plays an important role in cold-blooded animals and temperatures below or above the thermal limit can affect physiological functions, including adaptive and innate immunity, increase susceptibility to infection and even cause death. Therefore, how to prevent stress response and alleviate the harm caused by stress is currently one of the key subjects of research in this field. Anthraquinone extract contains emodin, chrysophenol, and rhein, and has been used as an immuno-stimulant and improves resistance to stress of fish.Megalobrama amblycephala, also known as the blunt snout bream or the Wuchang bream, is herbivorous and has been widely favored as a delicacy. It is the most important cultivated species of conventional freshwater fish in China. This species and other conventional freshwater fish, such as grass carp, common carp, black carp, and crucian carp, are commercially important for their value as food, In China, the production of M. amblycephala has been increasing in recent years and reached0.6million tons in2011.However, cultured M. amblycephala in China as well as in other parts of the world have suffered from serious disease problems caused by bacterial pathogens, and this has led to significant economic loss. Therefore, the development of non-chemical and natural therapeutics has become increasingly important. In order to study the mechanism of high temperature stress, our objective of research is to isolate and clone of cDNA sequence of heat shock protein60(HSP60) and heat shock protein90β (HSP90β), gain more biological information of HSP60and HSP90β and evaluate the gene expression of HSP60and HSP90in different tissue. The method is to measure the variation of immune parameters, antioxidative factors, blood hormone, HSPs expression of M. amblycephala under high temperature stress; to study effect of high temperature stress and recovery on the chronic and acute stress mechanism of M. amblycephala in aquarium with automatic temperature control breeding system with circle water; to study the mechanisms of how anthraquinone extracts can resist the infection of pathogenic bacteria under the acute and chronicle stress. We expect to set up a model of complex stress how to assess fish stress and find out the relationship between the physiological factors, HSPs expression and immunity of M. amblycephala under the stress of high temperature stress and verify the effects of anthraquinone extracts to prevent stress and resist the infection of pathogenic bacteria of M. amblycephala on the molecular basis, and further to provide theoretic basis for resistant stress in fish and to reduce the loss caused by stress in aquaculture.1Cloning and different tissue gene expression of HSP60and HSP90P of M. amblycephalaThis paper utilized RT-PCR and RACE(Rapid Amplification of cDNA Ends)methods and isolated and cloned for the first time the whole sequence of HSP60and HSP9Oβ of Megalobrama amblycephala. The result indicated the whole cDNA length of HSP60of M. amblycephalawas2253bp, including59bp5’ untranslated regions,1728bp open reading frames and466bp3’ untranslated regions containing Poly(A) signal AATAAA [excluding Poly(A)]. The open reading frame was totally coded575amino acids and calculated molecular weight61.24kDa. It’s identities were as high as88%-99%with theHSP60of other fishes, and70%-85%with the HSP60of Litopenaeus vannamei, Drosophila melanogaster and Homo sapiens.The result indicated the whole cDNA length of HSP90P of M. amblycephalawas2676bp, including89bp5’ untranslated regions,2253bp open reading frames and334bp3’ untranslated regions containing Poly(A) signal AATAAA [excluding Poly(A)]. The open reading frame was totally coded750amino acids and calculated molecular weight86.16kDa. It’s identities were as high as92%-97%with the HSP9Oβ of other fishes, and78%-80%with the HSP90β of Metapenaeus ensis, Drosophila melanogaster. We detected the gene expression of HSP60, HSP70, HSP90P in different tissue and the result showed that gene expression of HSP60, HSP70, HSP90β were highest in heart and brain and lowest in liver, the middle level in kidney and spleen. 2Effect of acute and chronic stress on serum biochemical, immunity and HSPs gene expression of M.amblycephalaThe fish (body weight:77±2.18g) were randomly divided into two groups. One is the control group (25±1℃), the other is the treatment group (32±1℃). Each group is divided into6repetition experiments. The glucose (GLU), cholesterol (CHOL), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), completement3(C3), completment4(C4), lysozyme (LSZ), cortisol (COR), adrenocorticotropic hormone (ACTH), Insulin, triiodothyronine (T3), thyroxine (T4) concentrations in the serum and anti-superoxide anion free radical (ASAFR),superoxide dismutase (SOD), malondialdehyde (MDA), HSP60, HSP70, HSP90mRNA level in liver were determined at Oh,2h,6h,12h after the high temperature stress. The results showed that compared with the control group(25±℃), the treatment group(32±℃) was associated with increased serum GLU, AST, ACTHCOR、T3、T4, hepatic MDA and HSP60, HSP70and HSP90gene expression, with decreased in serum C3, C4, GLB, CHOL, TG, Insulin and hepatic SOD or ASAFR content in the high stress group. Furthermore, the treatment group(32±℃) affect the ultrastructure of live cell.There were correlation between hepatic ASFER, SOD, MDA content, HSP60, HSP70, HSP90β gene expression and the serum G1U, COR, T3, T4, Insulin, AST, C3, C4. All the parameters play important roles in the high temperature stress. However the mechanism of high temperature stress need to be studied further. So it is suggested that high temperature stress may exert influence on the physiological function, improve the mRNA level of HSP60, HSP70, HSP90β in liver and impact on the non-specific immune ability of Wuchang bream.3The effect of recovery after high temperature stress on serum biochemical, immunity and HSPs gene expression of M.amblycephalaThe90healthy fish were acclimated for15days and randomly divided into6replication experiments. All fish were exposed by32℃high temperature stress for1day and recovered by25℃temperature for2days, then exposed by32℃high temperature stress for1day and recovered by25℃temperature for12days in the temperature controlled aquariums. The GLU, CHOL, TG, AST, ALT, C3, C4, LSZ, COR, ACTH, T3, T4concentrations in the serum andASAFR, SOD, MDA, HSP60, HSP70, HSP90β mRNA level in liver were determined at Oh,2h,6h,12h after the high temperature stress. The results showed that the serum and liver parameters became the trend of increasing or decreasing after the stress and recovery. After2d recovery after the first stress of32℃, the serum content of TG, ALB, C3, ALP, ACTH, Insulin, T3, T4and hepatic concentration of ASFER, SOD, MDA, HSPs reached the pre-stress level and AST, CHOL, TP, GLB, C4, GLU, COR didn’t recover the pre-stress level. After5d recovery after the second stress of32℃, the serum content of GLU, AST, TG, GLB, ALB, CHO, LTP, C3, C4, ALP, ACTH, Insulin and hepatic concentration of ASFER, SOD, MDA reached the pre-stress level and the serum COR, T3, T4only didn’t recover the pre-stress level. After12d recovery after the second stress of32℃, the serum content of GLU, AST, TG, GLB, TP, C3, C4, ALP, ACTH, Insulin, T3, T4and hepatic concentration of ASFER, SOD, MDA reached the pre-stress level and the serum ALB, CHOL, COR, T4didn’t recover the pre-stress level. So the high temperature stress has some influence on the normal physiological function of Wuchang bream, such as immunity and mortality of pathogenic infection and so on. Meanwhile, it also induce the increase of mRNA level of HSP60, HSP70, HSP90β.During the recovery period, some indices have reached the normal level5d recovery after stress, but the level of COR and T4increased, this maybe indirectly showed that COR plays some role in the recovery after acute high temperature stress.4Comparison study of the effects of anthraquinone extract and emodin from Rheum officinale Bail on the prevention of high temperature stress and disease resistance of M.amblycephalaFish were randomly divided into three groups:one was the control group, fed with a basal diet, and the other two were the treatment groups, fed with the basal diet supplemented with0.1%anthraquinone extract or60mg/kg emodin. After8weeks, fish were exposed to high temperature stress (34℃) for12hours. The serum cortisol, lysozyme, ALP, AST, ALT, glucose, total protein, TG, CHOL, hepatic catalase (CAT), SOD and MDA and relative level of hepatic HSP70mRNA were investigated before and after the stress. The results showed that compared with the control group, the doses of0.1%anthraquinone extract and60mg/kg emodin significantly increased serum lysozyme activities, SOD activities and the relative HSP70mRNA levels of liver and reduced serum ALT concentration before stress. After12h high temperature stress, the dose of0.1%anthraquinone extract group significantly increased serum lysozyme activity6h after stress, ALP activity2h after stress, total protein6h after stress, TG contents6h after stress, liver SOD activity2,6h after stress and the relative HSP70mRNA levels of liver2h after stress and reduced serum cortisol content2,12h after stress, AST12h after stress and ALT activity2,12h after stress, glucose content2h after stress, liver MDA content2,6h after stress compared with the control group. Moreover,60mg/kg emodin group significantly increased serum TG content6h after stress, liver CAT activity6h after stress, liver SOD activities2,6,12h after stress and the relative HSP70mRNA level of liver2,6h after stress and reduced serum cortisol content2,6,12h after stress, AST activity12h after stress, glucose content6h after stress, MDA content6h after stress compared with the control group. Artificial infection with Aeromonas hydrophila showed that the cumulative mortality in the control was about100%, while the cumulative mortality in the treatment group supplemented with0.1%anthraquinone extract was about83.33%and in the group supplemented with67.5mg/kg emodin was about80.00%. The results suggest that ingestion of supplements with0.1%anthraquinone extract and60mg/kg emodin can increase immune ability, enhance resistance against high temperature stress and A. hydrophila infection.5Comparison study of the effects of anthraquinone extract and emodin from Rheum officinale Bail on the growth, prevention of crowding stress of M. amblycephalaFish were randomly divided into three groups:one was the control group, fed with a basal diet, and the other two were the treatment groups, fed with the basal diet supplemented with0.1%anthraquinone extract or60mg/kg emodin. After8weeks, fish were exposed to crowding stress for1d. The growth, the serum cortisol, lysozyme, ALP, AST, ALT, glucose, hepatic CAT, SOD and MDA and relative level of hepatic HSP70mRNA before and after the stress were investigated. The results showed that compared with the control group, the doses of0.1%anthraquinone extract and60mg/kg emodin significantly increased weight gain rates, specific growth rates, serum lysozyme activities, SOD activities and the relative HSP70mRNA levels of liver and reduced the feed conversion ratios, serum cortisol and ALT concentration before stress. After1d crowding stress, the dose of0.1%anthraquinone extract group significantly increased serum ALP activity, SOD activities12,24h after stress and the relative HSP70mRNA levels of liver12,24h after stress and reduced serum cortisol contents12,24h after stress, glucose content12h after stress, AST and ALT activities12h after stress compared with the control group.Moreover,60mg/kg emodin group significantly increased serum lysozyme activity12h after stress, SOD activities12,24h after stress and the relative HSP70mRNA levels of liver12,24h after stress and reduced serum cortisol contents12,24h after stress, AST activity12h after stress, ALT activities12,24h after stress compared with the control group. The results suggest that ingestion of supplements with0.1%anthraquinone extract and 60mg/kg emodin can enhance resistance against crowding stress and promote the fish growth.6Effects of anthraquinone extract from Rheum officinale Bail on the physiological responses and HSP70gene expression of Megalobrama amblycephala under Aeromonas hydrophila infectionFish were randomly divided into threeroups:one was the control group, fed with a basal diet, and the other two were the treatment groups, fed with the basal diet supplemented with0.1%anthraquinone extract or60mg/kg emodin for8weeks. We then challenged the fish with A. hydrophila and recorded mortality and changes in serum cortisol, lysozyme, ALP, total protein, AST, ALT and hepatic CAT, SOD, MDA and the relative expression of HSP70mRNA. Supplementation with0.1%anthraquinone extract significantly increased serum lysozyme activity before infection, serum ALP activity at24h after infection, serum total protein concentration12h after infection, hepatic CAT activity12h after infection, hepatic SOD activity before infection, and the relative expression of hepatic HSP70mRNA both before infection and6h after infection.60mg/kg emodin significantly increased serum lysozyme activity before infection and6h,48h after infection, ALP activity24h,48h after infection, serum total protein concentration before infection and6h,12h after infection, CHOL content24h after infection, hepatic the relative expression of hepatic HSP70mRNA before stress.In addition, the supplemen of0.1%anthraquinone extract group had decreased levels of serum cortisol6h after infection, serum AST and ALT activities12h after infection, and hepatic MDA content12h after infection.60mg/kg emodin significantly had decreased levels of serum cortisol before infection, AST activity12h after infection, ALT activity and MDA content6h,12h after infection, GLU24h after infection. Mortality was significantly lower in the treatment group than the control. Our results suggest that ingestion of a basal diet supplemented with0.1%anthraquinone extract from R. officinale Bail and and60mg/kg emodin can enhance resistance against pathogenic infections in M. amblycephala.