Effects of Feeding Schizochytrium on Anti-Vibrio vulnificus Infection in Zebrafish
-
摘要:
目的 研究饲料中添加裂殖壶菌对鱼类抗细菌感染能力的影响，为裂殖壶菌作为饲料添加剂的应用提供理论基础。 方法 以6个月的成年斑马鱼为研究对象，以基础颗粒饲料为对照组（C组），试验组在基础饲料中添加干重3%的裂殖壶菌（S组），进行28 d饲喂试验。28 d后以创伤弧菌Vibrio vulnificus菌株FJ03-X2对斑马鱼进行攻毒，统计免疫保护率，并收集斑马鱼的肾脏组织提取RNA，逆转录成cDNA，通过Real-time RT-PCR检测相关基因lysozyme和tnfb的表达。 结果 饲料中添加3%裂壶菌能够显著提高斑马鱼感染创伤弧菌后的存活率，相对保护率为42.8%；Real-time RT-PCR检测的结果发现S组的斑马鱼肾脏中溶菌酶Lysozyme的mRNA水平极显著高于对照组（P＜0.01），而肿瘤坏死因子TNFb的mRNA水平则极显著低于对照组（P＜0.01）。 结论 裂殖壶菌作为营养添加剂可以提高斑马鱼抗创伤弧菌感染的能力。 Abstract:Objective A feeding experiment was conducted in order to study the effects of feeding Schizochytrium on the resistance of fish to bacterial infection. Methods Six-month adult zebrafishes were fed with diety added 3% Schizochytrium(Group S) for 28 days in a laboratory culture system, and the control with only basic diety(Group C). Then Vibrio vulnificus FJ03-X2 was applied for infection, and the relative survival percent of zebrafishes were analyzed. And RNA was extracted from the kidney tissues and reverse transcribed into cDNA to detect the experison of lysozyme and tnfb. Results The survival rate of Group S was significantly increased with immune protective rate reaching 42.8%. The mRNA level of Lysozyme in the kidney of zebrafish in Group S was significantly higher than that in the control (P＜0.01), while the mRNA level of TNFb was significantly lower than that in the control group (P＜0.01). Conclusion As a nutrient additive, Schizochytrium could improve the ability of zebrafish to resist bacterial infection, which provided a theoretical basis for the application of Schizochytrium in fishery industry. -
Key words:
- Schizochytrium /
- zebrafish /
- Vibrio vulnificus /
- feed additive
-
图 1 Real-time RT-PCR检测创伤弧菌感染后肾脏中lysozyme的mRNA水平
注：C为对照组攻毒前；S为试验组攻毒前；C+V.v-L为对照组创伤弧菌攻毒存活的斑马鱼；C+V.v-D为对照组创伤弧菌攻毒死亡的斑马鱼；S+V.v-L为试验组创伤弧菌攻毒存活的斑马鱼；S+V.v-D为试验组创伤弧菌攻毒死亡的斑马鱼；**表示试验组与对照组存在极显著性差异P＜0.01。图2同。
Figure 1. mRNA level of lysozyme in the kidney of zebrafish under infection by Vibrio vulnificus
Note: C-Zebrafish in the control group before challenge; S-Zebrafish in the experimental group before challenge; C+V.v-L-Survival zebrafish in the control group challenged by Vibrio vulnificus; C+V.v-D-Dead zebrafish in the control group challenged by Vibrio vulnificus; S+V.v-L- Survival zebrafish in the experimental group challenged by Vibrio vulnificus; S+V.v-D- Dead zebrafish in the experimental group challenged by Vibrio vulnificus; **-The value indicates significant difference，P＜0.01. The same as Fig. 2.
图 2 Real-time RT-PCR检测创伤弧菌感染后肾脏中tnfb的mRNA水平
Figure 2. mRNA level of tnfb in the kidney of zebrafish under infection by Vibrio vulnificus
表 1 基础饲料的营养成分
Table 1. Nutrient composition of basic feed
成分 Ingredient 含量 Content/% 粗蛋白质 Crude protein ≥40.0 粗纤维 Crude fiber ≤4.0 氨基酸 Amino acid ≥30.0 粗灰分 Crude ash ≤16.0 水分 Water content ≤11.0 盐分 Salt ≤3.0 总磷 Total phosphorus content ≥0.9 钙 Calcium ≥1.5 
下载: 导出CSV
表 2 PCR引物序列
Table 2. PCR primer sequence
目的基因 Objective gene 正向引物序列（5′-3′）Forward primer sequence（5′-3′） 反向引物序列（5′-3′）Reverse primer sequence（5′-3′） actb1 CACTTCACGCCGACTCAAAC TCGGGGATGCTTATTTGCCA tnfb GCCAACCCATTTCAGCGATTG GGCATGTGATGAAGCCAAACGAA lysozyme CCGTAGTCCTTCCCCGTATCA GATTTGAGGGATTCTCCATTGG
下载: 导出CSV
表 3 裂殖壶藻ATCC MYA-1381的脂肪酸组成
Table 3. Fatty acid composition of Schizochytrium ATCC MYA-1381
脂肪酸 Fatty acid 百分比 Percentage% C14∶0 3.81 C15∶0 0.95 C16∶0 43.22 C16∶1n7c 0.16 C17∶0 0.38 C17∶1n7c 0.13 C18∶0 0.74 C18∶1n9c 0.08 C18∶2n6c 0.04 C18∶3n3c 0.07 C20∶0 0.19 C20∶3n6c 0.67 C20∶4n6c 0.17 C20∶5n3c 1.25 C23∶0 0.08 C24∶0 0.08 C22∶6n3c 39.62 其他 Others 8.35
下载: 导出CSV
表 4 斑马鱼创伤弧菌攻毒试验
Table 4. Infection test of zebrafish with Vibrio vulnificus
组别 Groups 攻毒尾数 Total number 死亡尾数 Dead number 死亡率 Death rate（24 h） 免疫保护率 Relative survival percent（RPS） 对照组 Control group 15 7 46.7% — 试验组 Experimental group 15 4 26.7% 42.8%
下载: 导出CSV
-
[1] STROBEL C, JAHREIS G, KUHNT K. Survey of n-3 and n-6 polyunsaturated fatty acids in fish and fish products [J]. Lipids in Health and Disease, 2012, 11(1): 144. doi: 10.1186/1476-511X-11-144 [2] BELL J G, HENDERSON R J, TOCHER D R, et al. Substituting fish oil with crude palm oil in the diet of Atlantic salmon(Salmo salar) affects muscle fatty acid composition and hepatic fatty acid metabolism [J]. The Journal of Nutrition, 2002, 132(2): 222−230. doi: 10.1093/jn/132.2.222 [3] 李美玉, 李健, 陈萍, 等. 维生素E和裂壶藻对中国对虾生长及TLR/NF-κB表达水平的影响 [J]. 中国渔业质量与标准, 2012, 2(2):37−44.LI M Y, LI J, CHEN P, et al. Effects of VE and Schizochytrium on growth and TLR / NF -κB expression level of Fenneropenaeus chinensis [J]. China Fishery Quality And Standards, 2012, 2(2): 37−44.(in Chinese) [4] 朱路英, 张学成, 王淑芳, 等. 一种海洋真菌: 裂殖壶菌的营养成分分析 [J]. 食品科学, 2009, 30(24):272−275. doi: 10.3321/j.issn:1002-6630.2009.24.058ZHU L Y, ZHANG X C, WANG S F, et al. Analysis of nutritional components of a marine fungus: Schizochytrium limanium [J]. Food Science, 2009, 30(24): 272−275.(in Chinese) doi: 10.3321/j.issn:1002-6630.2009.24.058 [5] CALDER P C. N-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases [J]. The American Journal of Clinical Nutrition, 2006, 83(6): 1505S−1519S. doi: 10.1093/ajcn/83.6.1505S [6] SADEGHI S, WALLACE F A, CALDER P C. Dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice [J]. Immunology, 1999, 96(3): 404−410. doi: 10.1046/j.1365-2567.1999.00701.x [7] HAYASHI N, TASHIRO T, YAMAMORI H, et al. Effects of intravenous ω-3 and ω-6 fat emulsion on cytokine production and delayed type hypersensitivity in burned rats receiving total parenteral nutrition [J]. Journal of Parenteral and Enteral Nutrition, 1998, 22(6): 363−367. doi: 10.1177/0148607198022006363 [8] KELLEY D S, DAUDU P A. Fat intake and immune response [J]. Progress in Food & Nutrition Science, 1993, 17(1): 41−63. [9] SAWYER M B, FIELD C J. Possible mechanisms of ω-3 PUFA anti-tumour action[C]//Dietary Omega-3 Polyunsaturated Fatty Acids and Cancer. Dordrecht: Springer Netherlands, 2009: 3-38. [10] FARMER J J III. vibrio (" beneckea”) vulnificus, the bacterium associated with Sepsis, septicæmia, and the Sea [J]. The Lancet, 1979, 314(8148): 903. [11] BIOSCA E G, AMARO C, ESTEVE C, et al. First record of Vibrio vulnificus biotype 2 from diseased European eel, Anguilla anguilla L [J]. Journal of Fish Diseases, 1991, 14(1): 103−109. doi: 10.1111/j.1365-2761.1991.tb00581.x [12] 冯伟. 维生素C、E和裂壶藻对中国对虾非特异性免疫功能影响的研究[D]. 海: 上海海洋大学, 2011.FENG W. Effects of supplemental different level Vc, Ve and Schizochytrium on non-specific immunity of Chinese shrimp (Fenneropenaeus Chinensis)[D]. Shanghai: Shanghai Ocean University, 2011. (in Chinese) [13] 焦建刚, YUAN Kathy, THABUIS Clémentine, 等. 裂殖壶藻发酵粉对南美白对虾生长性能和肌肉营养成分的影响 [J]. 上海海洋大学学报, 2014, 23(4):523−527.JIAO J G, KATHY Y, THABUIS C, et al. The effect of dried algae Schizochytrium sp. on growth performance and nutrients in the muscle of white shrimp (Penaeus vannamei) [J]. Journal of Shanghai Ocean University, 2014, 23(4): 523−527.(in Chinese) [14] 黄亮华, 李浩洋, 李彬, 等. 裂壶藻对刺参生长、免疫及消化酶的影响 [J]. 渔业科学进展, 2014, 35(3):91−97. doi: 10.11758/yykxjz.20140313HUANG L H, LI H Y, LI B, et al. Effects of dietary Schizochytrium limacinum on the growth, immunity and digestion enzyme activity of sea cucumber Apostichopus japonicus [J]. Progress in Fishery Sciences, 2014, 35(3): 91−97.(in Chinese) doi: 10.11758/yykxjz.20140313 [15] 许斌福, 林天龙, 董传甫. 鳗鲡创伤弧菌的分子鉴定 [J]. 中国人兽共患病杂志, 2005, 11(21):995−997.XU B F, LIN T L, DONG C F. Molecular identification of Vibrio vulnificus isolated from the diseased European eels in mainland China [J]. Chinese Journal of Zoonoses, 2005, 11(21): 995−997.(in Chinese) [16] 陈华, 李素一, 张丽娟, 等. CpG-ODN 2006对斑马鱼抗创伤弧菌感染的作用 [J]. 福建农业学报, 2018, 33(1):1−6.CHEN H, LI S Y, ZHANG L J, et al. Functions of CpG-ODN 2006 on Resistance of Zebra Fish against Vibrio vulnificus [J]. Fujian Journal of Agricultural Sciences, 2018, 33(1): 1−6.(in Chinese) [17] MILLER M R, NICHOLS P D, CARTER C G. Replacement of fish oil with thraustochytrid Schizochytrium sp. L oil in Atlantic salmon parr(Salmo salar L.) diets [J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007, 148(2): 382−392. [18] GANUZA E, BENÍTEZ-SANTANA T, ATALAH E, et al. crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to fisheries-derived oils from seabream(Sparus aurata) microdiets [J]. Aquaculture, 2008, 277(1/2): 109−116. [19] 吉红, 周继术, 曹福余, 等. DHA对鲤抗氧化能力影响的初步研究 [J]. 上海海洋大学学报, 2009, 18(2):2142−2149.JI H, ZHOU J S, CAO F Y, et al. Effect of DHA on anti-oxidation capacity in common carp(Cyprinus carpio L.) [J]. Journal of Shanghai Ocean University, 2009, 18(2): 2142−2149.(in Chinese) [20] 王桂芹, 牛小天, 闫先春, 等. 饲料中添加维生素E和二十二碳六烯酸对鲤鱼生长和抗病力的影响 [J]. 上海交通大学学报(农业科学版), 2010, 28(2):124−128.WANG G Q, NIU X T, YAN X C, et al. Effect of dietary vitamin E and DHA levels on growth and disease resistance of Cyprinus carpio juvenile [J]. Journal of Shanghai JiaoTong University(Agricultural Science), 2010, 28(2): 124−128.(in Chinese) [21] 刘然然. 大鲵c-型和g-型溶菌酶基因的生物信息学分析和表达研究[D]. 杨凌: 西北农林科技大学, 2018.LIU R R. Bioinformatics analysis and expression research of c-style lysozyme gene and g-style lysozyme gene from Chinese giant salamander[D]. Yangling, China: Northwest A & F University, 2018. (in Chinese) [22] 邱本丹. 大黄鱼溶菌酶对鳗弧菌胁迫的响应研究[D]. 舟山: 浙江海洋大学, 2016.QIU B D. The response of c-type and g-type lysozyme in large yellow croaker(Larimichthys crocea) to Vibrio anguillarum[D]. Zhoushan: Zhejiang Ocean University, 2016. (in Chinese) [23] PFEFFER K. Biological functions of tumor necrosis factor cytokines and their receptors [J]. Cytokine & Growth Factor Reviews, 2003, 14(3/4): 185−191. [24] 胡梦平, 刘巧刿. 肿瘤坏死因子-α在铜绿假单胞菌肺炎中的变化及意义 [J]. 海南医学院学报, 2009, 15(8):864−866. doi: 10.3969/j.issn.1007-1237.2009.08.013HU M P, LIU Q G. The role of tumor necrosis factor alpha in Pseudomonas aeruginosa pneumonia [J]. Journal of Hainan Medical University, 2009, 15(8): 864−866.(in Chinese) doi: 10.3969/j.issn.1007-1237.2009.08.013 [25] CHENG C L, HUANG S J, WU C L, et al. Transgenic expression of Omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection [J]. Journal of Biomedical Science, 2015, 22: 103. doi: 10.1186/s12929-015-0208-1 -
点击查看大图
图(2) / 表(4)
计量
- 文章访问数: 1447
- HTML全文浏览量: 634
- PDF下载量: 28
- 被引次数: 0
