Effects of Feeding Schizochytrium on Anti-Vibrio vulnificus Infection in Zebrafish
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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
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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 
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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
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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
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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%
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