| This study explored the copper nutrition physiology of M. nipponense by using comprehensive experiment technology of nutrition, physiology, biochemistry and molecular biology. These included dietary copper requirement of juvenile M. nipponense, the effects of dietary copper level on the growth, antioxidant ability and immune response under normal and nitrite stress conditions, the effects of dietary copper and zinc on prawn. In the same time, containing-copper proteins, such as hemocyanin and metallothionein were cloned and evaluated how their expressions were regulated by dietary copper and zinc levels.1. Dietary copper requirement of juvenile M. nipponenseThe present experiment evaluated the effects of dietary copper (Cu) levels on the growth, antioxidant and immune enzymes activities, resistence to A. hydrophila infection of juvenile oriental river prawn M. nipponense, and the dietary copper requirement was determined. Seven semi-purified diets with graded levels of copper (2.8,12.2,20.9,29.8,43.1,78.9and157.1mg kg-1diet) as CuSO4·5H2O were fed to juvenile prawn (initial weight0.101±0.002g) for56days. These results showed:(1) The prawns fed with2.8-78.9mg Cu kg-1diet obtained higher weight gain and lower feed conversion ratio than157.1mg Cu kg-1group (P<0.05). Cu concentrations in hepatopancreas, muscle and whole body in prawn tended to increase with increased dietary Cu concentration (P<0.05);(2) Activities of hepatopancreas Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GPx) and total antioxidant capacity (T-AOC) were the highest (P<0.05) in the43.1mg Cu kg-1group. The hepatopancreas malondialdehyde (MDA) content was lower (P<0.05) in the prawns fed with29.8mg Cu kg-1than that in the prawns fed with2.8,12.2,78.9 and157.1mg Cu kg-1diet.(3) The diets supplemented with43.1and78.9mg Cu kg-1significantly increased activities of plasma alkaline phosphatase (AKP), acid phosphatase (ACP)(P<0.05). The serum lysozyme (LYZ) activity elevated with the increase of dietary Cu level, and peaked at43.1mg Cu kg-1diet, then exhibit a decreasing trend (P<0.05). After the56days feeding experiment, prawns were challenged with A. hydrophila by injection. The cumulative mortalities of the prawns fed with20.9-43.1mg Cu kg-1diet were significantly lower than that of the prawns fed with2.8and157.1mg Cu kg-1.(4) The optimum requirement of dietary Cu in juvenile prawn was estimated as26.9and27.8mg kg-1diet based on the cumulative mortality rate and whole-body Cu retention respectively. This study indicated that deficient or excess dietary Cu could induce oxidative stress, affect prawn antioxidant ability, and decrease prawn immunity.2. The effect of dietary copper level on the expression of hemocyanin in M. nipponenseFor prawn, the hemocyanin also is a copper-containing protein. Besides as an oxygen carrier, it also possesses immune function. In order to better understand the significant functions of copper to M. nipponense, we explored the effect of dietary copper level on the hemocyanin expression. In this study, a hemocyanin subunit designated as MnHc-1was cloned and characterized from the oriental river prawn by using rapid amplification of the cDNA ends (RACE) approach. The full length cDNA of MnHc-1was2163bp, containing a2028bp open reading frame (ORF) encoding a polypeptide of675amino acids with a calculated molecular weight of78.060kDa and an isoelectric point of5.45. A putative signal peptide of21amino acids was found in the N-terminus. Homology analysis showed that MnHc-1deduced amino acid had75%,73%,70%and68%similarity to that of the A. moluccensis gamma subunit, P. monodon, C. quadricarinatus and E. sinensis, respectively. The result of phylogenetic analysis revealed that MnHc-1was more closely related to the gamma subunit of hemocyanin in Decapoda. Quantitative real-time RT-PCR analysis showed that the MnHc-1mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary and stomach, with the highest level in the hepatopancreas. In the A. hydrophila challenge trial, the MnHc-1mRNA transcripts in the hemocytes were significantly down-regulated at3h post-injection, then up-regulated at6h and12h, and followed by a gradual recovery from24to48h. The levels of MnHc-1mRNA expression in the hepatopancreas and oxyhemocyanin content in hemolymph were also analyzed after M. nipponense were fed seven diets containing2.8,12.2,20.9,29.8,43.1,78.9and157.1mg Cu kg-1diet for56days, respectively. The level of MnHc-1mRNA was significantly higher in the prawns fed43.1-157.1mg Cu kg-1diet than that fed2.8-29.8mg Cu kg-1diet. The oxyhemocyanin content elevated with the increase of dietary Cu level, and peaked at43.1mg Cu kg-1diet, then exhibit a decreasing trend (P<0.05). This study illustrates that the hemocyanin expression can be affected by dietary copper, and the hemocyanin may be potentially participated in the anti-bacterial defense of M. nipponense.3. The effects of dietary Cu and Zn on the growth, antioxidant ability and immune response in M. nipponenseMicro-minerals copper and zinc share similar physicochemical property, some researchers have paid specific attentions on the interaction between copper and zinc, and based on the physiological importance of copper to M. nipponense, we explored the effects of dietary copper and zinc levels on the growth, minerals contents, antioxidant and immune enzymes activities of juvenile M. nipponense. Nine semi-purified diets with three levels of copper (0,30and180mg kg-1diet, low Cu, moderate Cu and high Cu) and three levels of zinc (0,35and210mg kg-1diet, low Zn, moderate Zn and high Zn) were fed to juvenile prawn (initial weight0.103±0.002g) in five replicates for56days. Moderate dietary Cu or Zn significantly increased prawn WG and SGR (P<0.05), but not survival rate (P>0.05). Whole body and hepatopancreas Cu or Zn contents elevated with the increase of dietary Cu or Zn level. High dietary Zn decreased prawn hepatopancreas Cu content. Diets supplemented with moderate Cu or moderate Zn or high dietary Zn all could significantly increase Cu-Zn SOD, GSH-Px and T-AOC activities, and significantly decreased MDA content. In high Cu groups, high Zn could increase prawns antioxidant enzymes activities, decrease MDA content. Serum AKP activity in prawns elevated with the increase of dietary Zn level regardless dietary Cu level. Low dietary Cu significantly suppressed AKP activity. Moderate dietary Cu significantly increased serum LYZ and PO activities in M. nipponense regardless of dietary Zn level. LYZ and PO activities were higher in prawns fed moderate or high Zn than prawns fed low dietary Zn. The present study indicated that dietary Cu and Zn levels both can affect growth, antioxidant activity and immune response in M. nipponense, and the interaction between Cu and Zn was found in antioxidant ability and immunity.4. The effects of dietary Cu and Zn on the expression of metallothionein in M. nipponenseMetallothionein is a kind of metal binding protein with low molecular weight, can be induced by minerals such as copper or zinc, and also can bind with copper or zinc. In order to better understand the copper physiology of nutrition in M. nipponense, we evaluated the effect of dietary copper and zinc levels on metallothionein expression. In this study, a metallothionein designated as Mn-MT was cloned and characterized from the oriental river prawn by using rapid amplification of the cDNA ends (RACE) approach. The full length cDNA of Mn-MT was665bp, containing a180bp open reading frame (ORF) encoding a polypeptide of59amino acids with a calculated molecular weight of6.085kDa and an isoelectric point of7.73. The protein contained a high percentage of cysteine residues (30.5%) and followed by lysine (16.95%), serine (10.17%) and proline (8.47%). Homology analysis showed that Mn-MT deduced amino acid had78%,75%and75%similarity to that of the H. americanus, P. monodon and E. sinensis respectively. Quantitative real-time RT-PCR analysis showed that the Mn-MT mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, muscle and stomach, with the highest level in the hepatopancreas. The levels of Mn-MT mRNA expression in the hepatopancreas were analyzed after M. nipponense were fed four diets supplemented with0,20,40and160mg Cu kg-1diet for56days, respectively. The level of Mn-MT mRNA was increased with the increase of dietary Cu level, and peaked at40mg Cu kg-1diet group, then exhibit a decreasing trend. The Mn-MT mRNA expression in the hepatopancreas were also analyzed after M. nipponense were fed nine diets supplemented with three levels Cu (0,30and180mg kg-1; low Cu, moderate Cu and high Cu) and three levels Zn (0,35and210mg kg-1; low Zn, moderate Zn and high Zn) for8weeks, respectively. Mn-MT mRNA expression in prawns fed high Zn were significantly higher than prawns fed moderate or low Zn regardless of dietary Cu level, moderate Zn group was higher than low Zn group, but was not significant. This study indicated that the Mn-MT transcription exhibited different dose-dependent response to dietary Cu and Zn; metallothionein induced by high Zn bind to Cu maybe decrease available Cu.5. Effects of dietary copper and zinc levels on hemoycanin expression and resistance to hypoxia stress in M. nipponenseThe effect of dietary copper and zinc on hemocyanin expresson in M. nipponense was analyzed. After the56days feeding experiment, prawns were stressed with2.5mg L"1dissolved oxygen for12h. Prawns fed with moderate Cu had the highest Mn-Hcland Mn-Hc2mRNA expression and oxyhemocyanin content, the lowest values were found in low Cu groups (P<0.05). Dietary high Zn significantly decreased oxyhemocyanin content, Mn-Hc1and Mn-Hc2mRNA expression (P<0.05). Prawns fed with moderate dietary Cu or Zn had higher Mn-CcO mRNA expression than other groups. After stress, the highest hemocyanin transcription expression was found in prawns fed with high Cu (P<0.05), and was higher than before stress. Prawns fed with low Zn had lower hemocyanin mRNA expression than the prawns fed diets with moderate or high Zn levels (P<0.05). After stress, the oxyhemocyanin content was higher than before stress except prawns fed with low dietary copper and low zinc. It follows that hemocyanin expression was closely related to dietary copper level, storage of copper was mobilized to synthesis hemocyanin in hypoxia stress.6. Effect of dietary copper levels on M. nipponense under nitrite exposureBased on the effect of dietary copper on hemocyanin expression and the reduction of hemocyanin affinity to oxygen under nitrite exposure, so we hypothesized that supplement dietary copper can alleviate the effect of nitrite on M. nipponense. Under two levels of nitrite (low nitrite:0.01mg L-1;high nitrite:1.90mg L-1), four semi-purified diets supplemented with graded levels of copper (0,30,60and180mg kg-1diet) as CUSO4·5H2O were fed to juvenile prawn (initial weight0.103±0.003g) for56days. Growth, antioxidant ability and immunity of prawns with low nitrite were higher than those of prawns with high nitrite. Prawns fed with30and60mg Cu kg-1diet had significantly higher WG, SGR, antioxidant and immune enzymes activities than those prawns fed diets with copper deficiency and excess regardless of nitrite level (P<0.05). MDA was the lowest in prawns fed diets supplemented with30mg Cu kg-1and the highest in180mg kg-1group (P<0.05) regardless of nitrite level. Higher nitrite significantly decreased hepatopancreas hemocyanin and cytochrome c oxidase transcription expressions in M. nipponense, but significantly increased hemolymph oxyhemocyanin content (P<0.05). The highest hemocyanin and cytochrome c oxidase mRNA expressions and oxyhemocyanin content were found in prawns fed60mg Cu kg-1diet group (P<0.05). After the56days feeding experiment, prawns were stressed with hypoxia of2.5mg1-1dissolved oxygen for12h. After hypoxia stress, oxyhemocyanin of all groups and hemocyanin mRNA expression of180mg Cu kg-1diet group increased. Hemocyanin transcription expression in prawns fed60and180mg Cu kg-1diet was significantly higher than other group prawns and the highest oxyhemocyanin content was in group of60mg Cu kg-1diet regardless of nitrite level (P<0.05). The present study indicated that nitrite stress could inhibit growth, immunity and resistance to hypoxia in prawn, but supplemented30-60mg Cu kg-1diet could alleviate these adverse effects of nitrite. |
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