碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 95 / The objective of this study is aimed to comprehend the molecular characterization and expression of ferritin gene, and to monitor the survival, immune and physiological responses, and the expression of haemocyte ferritin of the white shrimp, Litopenaeus vannamei under Vibrio and WSSV challenge for 72 hr period. Correlations between physiological and immunological parameters, involvements and physiological roles of ferritin in the immunological compensation process were further attempted.
Ferritin, a cytosolic iron storage protein composed of 24 subunits of heavy chain and light chain, is an intracellular protein primarily involved in iron metabolism and detoxification. A full-length ferritin cDNA was cloned from the haemocyte of the Pacific white shrimp. It comprises 1249 bp, including 132 bp in the 5’-untranslated region, 510 bp in the open reading frame which encodes 170 amino acid residues, and 607 bp in the 3’-untranslated region. The ferritin sequence has been registered in the GenBank (AY955373). Alignments of the deduced amino acid sequence further showed that the Pacific white shrimp ferritin is evolutionally closer to crayfish, tick and brine shrimp than others, sharing 86%, 79% and 78% identity, respectively. The tissue-specific expression pattern was examined by reverse transcript polymerase chain reaction and real-time quantitative PCR. The ferritin mRNA is expressed in various tissues of the shrimp, highest in haemocyte.
The susceptibilities of the white shrimp to the challenge of Vibrio alginolyticus and white spot syndrome virus (WSSV) were determined, and the physiological and immune responses of these shrimp were also monitored for 72 hr period. The immune parameters measured include total haemocyte count, phenoloxidase and superoxide dimutase activities and respiratory burst, while the physiological parameters cover hemolymph glucose, lactate and lipid contents.
The mortality of the white shrimp started to appear at 12 hr after Vibrio and WSSV challenge, and depression of the immune parameters (total haemocyte count, phenoloxidase and superoxide dimutase activities, and respiratory burst) were also observed. The expression of ferritin gene was up-regulated in both cases. Besides, occurrence of the minimum level of immune parameters under the challenge varied with types of pathogens, 36 hr under Vibrio and 12~24 hr under WSSV. The physiological parameters (hemolymph glucose, lactate and lipid) all notably elevated under Vibrio and WSSV challenge, and the time of highest increases varied also with pathogens. These parameters were all peaked at 36 hr under Vibrio infection, while under WSSV challenge, hyperglycemia occurred at 24 hr, hyperlacticemia at 12 hr and hyperlipidemia at 24 hr. These observations suggested that the pathogen challenge imposed physiological stress and immune depression of the white shrimp, resulted in increased mortality. In addition, the handling and injection stress do induce increasing trends of the physiological parameters, and the challenge from the pathogens augmented the magnitude of elevation, but this was not true for the immune parameters.
Effects of a single ferritin injection at the doses of 0.1, 0.5 and 1 ng ferritin/g shrimp on the physiological and immune responses were also monitored for 72 hr period. The experiment with WSSV challenge was also attempted. The administration of ferritin improved the immunity of the white shrimp, indicated by significant elevation of immune parameters to the control level or even higher, and the survival was improved as well. The best survival of 87.5% was obtained by a single injection of 0.5 ng ferritin/g shrimp, at this dose, the immunity was notably enhanced. It is therefore concluded that ferritin possess the immunostimulant functions, indicated by improved resistance to WSSV and increased survival under the challenge. Furthermore, the physiological parameters were not affected by ferritin injection, even in the case that the shrimp were challenged with WSSV. It is evidently demonstrated that ferritin is able to reduce stress response and is consequently capable to maintain physiological homeostasis. The ferritin does play important roles in the innate immunity of the white shrimp.
Key words: Ferritin, Litopenaeus vannamei, immune parameters, physiological parameter, pathogen challenge, Vibrio alginolyticus, white spot syndrome virus (WSSV)
| Identifer | oai:union.ndltd.org:TW/095NTOU5111029 |
| Date | January 2007 |
| Creators | Yuan-Hwa Ruan, 阮燕華 |
| Contributors | Chin-Hwa Hu, Chin-Ming Kuo, 胡清華, 郭欽明 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 128 |
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