Microbial analysis of Litopenaeus vannamei during partial freezing storage by Illumina high throughput sequencing
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摘要: 文章以微冻贮藏的凡纳滨对虾 (Litopenaeus vannamei) 整虾、去头尾虾和虾仁的表面微生物作为研究对象，采用高通量测序技术鉴定不同贮藏时期样品菌群的组成及变化情况。结果显示：1) 各组样品的操作分类单元 (Operational taxonomic unit, OTU) 随贮藏时间的推移呈下降趋势，贮藏4周后虾仁OTU数量最少，整虾与去头尾虾OTU数量相对较多；2) 各组样品的赵氏指数 (Chao)、艾斯指数 (ACE) 和香农指数 (Shannon) 逐渐减小，辛普森指数 (Simpson) 增加，即贮藏过程中样品菌群丰度和多样性逐渐降低；3) 在属水平上，弧菌属 (Vibrio) 是各组样品的最初优势菌，弓形菌属 (Arcobacter) 在去头尾虾和整虾中存在较多。微冻4周后，假单胞菌属 (Pseudomonas)、希瓦氏菌属 (Shewanella) 和嗜冷杆菌属 (Psychrobacter)成为优势腐败菌；4) 属水平上的热图和关键物种分析显示，3种形式的对虾菌种组成和比例有所差异。综上，凡纳滨对虾贮藏时间和形态不同，其表面微生物种类、丰度等差异显著，在冷链流通过程中需要针对性地抑制微生物优势以保障产品品质。Abstract: Taking Litopenaeus vannamei of different forms (whole shrimp, decapitated shrimp, shrimp meat) as research objects, we applied Illumina high throughput sequencing technology to identify the surface microbial composition and changes of samples during partial freezing storage. The results show that: 1) The operational taxonomic units (OTUs) number of all samples decreased with the extension of storage time. After four weeks of storage, the whole shrimp and the decapitated shrimp had more OTUs, and the OTU number in shrimp meat was the least. 2) Chao, ACE and Shannon indices of all samples decreased but Simpson index increased, which indicates that the abundance and diversity of samples reduced during the storage. 3) At genus level, Vibrio was the main microorganism of all samples at the beginning, and Arcobacter existed more in the decapitated shrimp and the whole shrimp. After four weeks of storage, Pseudomonas, Shewanella and Psychrobacter became the dominant spoilage bacteria. 4) The heatmap and key species analysis at genus level show that the composition and proportion of three forms of shrimp samples were different. Therefore, different forms of L. vannamei had different microbial compositions and abundance in various storage periods. It is necessary to inhibit the advantages of microorganisms specifically to ensure the quality of the products during the cold chain process.
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图 1 不同形式凡纳滨对虾的韦恩图
R. 虾仁样品；Q. 去头尾虾样品；Z. 整虾样品
Figure 1. Venn diagram of L. vannamei with different forms
R. Shrimp samples; Q. Decapitated shrimp samples; Z. Whole shrimp samples
图 2 不同形式凡纳滨对虾多样性指数的稀释曲线
Figure 2. Rarefaction curves of Alpha diversity indices of L. vannamei with different forms
图 3 不同形式对虾样品微生物物种组成 (目水平)
r. 虾仁样品；q. 去头尾虾样品；z. 整虾样品；数字0、2、4分别表示贮藏0周、2周和4周；后图同此
Figure 3. Bacterial community composition at Order level of L. vannamei with different forms
r. Shrimp meat; q. Decapitated shrimp; z. Whole shrimp; the number 0, 2 and 4 indicate the storage time of 0 week, 2 weeks and 4 weeks. The same below.
图 4 不同形式对虾样品微生物物种组成 (属水平)
Figure 4. Bacterial community composition at genus level of L. vannamei with different forms
图 5 不同形式对虾样品微生物丰度聚类热图 (属水平)
同一行中颜色深浅表示该微生物在不同样品中的丰度差异；图上方进化树表明样本间的相似程度；图左方进化树表明微生物间的相似程度
Figure 5. Microbial community heatmap analysis at genus level of L. vannamei with different forms
The shade of color in the same row indicates the difference in the abundance of the microorganism in different samples; the evolutionary tree on the top of the figure indicates the degree of similarity between samples; the evolutionary tree on the left of the figure indicates the degree of similarity between microorganisms.
表 1 不同形式凡纳滨对虾的测序覆盖率和操作分类单元数量
Table 1. Coverage and OTU number of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp覆盖率
Coverage操作分类单元数量
OTU number覆盖率
Coverage操作分类单元数量
OTU number覆盖率
Coverage操作分类单元数量
OTU number0周 0 week 0.998 7 281 0.998 4 281 0.998 8 316 1周 1 week 0.998 5 189 0.999 0 401 0.999 1 322 2周 2 weeks 0.999 2 113 0.998 8 297 0.998 8 333 3周 3 weeks 0.999 9 36 0.999 8 50 0.999 8 50 4周 4 weeks 0.999 9 31 0.999 8 54 0.999 7 59 
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表 2 不同形式凡纳滨对虾的Alpha丰富度指数表
Table 2. Alpha abundance indices of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp赵氏指数 Chao 艾斯指数 ACE 赵氏指数 Chao 艾斯指数 ACE 赵氏指数 Chao 艾斯指数 ACE 0周 0 week 341 338 342 336 375 378 1周 1 week 231 236 429 437 363 365 2周 2 weeks 138 143 382 377 397 401 3周 3 weeks 45 50 80 69 80 120 4周 4 weeks 42 48 61 70 96 112
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表 3 不同形式凡纳滨对虾的Alpha多样性指数表
Table 3. Alpha diversity indices of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp香农指数Shannon 辛普森指数Simpson 香农指数Shannon 辛普森指数Simpson 香农指数Shannon 辛普森指数Simpson 0周 (0 week) 2.61 0.152 1 2.96 0.099 7 3.42 0.061 2 1周 (1 week) 1.91 0.278 3 3.43 0.086 9 2.49 0.213 1 2周 (2 weeks) 1.50 0.357 5 2.84 0.104 3 3.08 0.094 4 3周 (3 weeks) 1.08 0.436 6 1.43 0.345 3 1.59 0.358 4 4周 (4 weeks) 1.06 0.446 2 1.23 0.480 9 1.41 0.361 5
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表 4 不同形式对虾样品关键物种差异 (属水平)
Table 4. Key species differences at genus level of L. vannamei with different forms
% 前10个物种
Top 10 species虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp假单胞菌属 Pseudomonas 36.730 4 35.589 3 15.183 3 希瓦氏菌属 Shewanella 33.741 4 27.325 8 11.265 6 嗜冷杆菌属 Psychrobacter 0.339 6 8.251 5 31.040 7 弧菌属 Vibrio 13.798 0 4.175 6 2.838 0 不动杆菌属 Acinetobacter 0.745 0 2.723 2 4.240 6 黄杆菌属 Flavobacterium 0.363 5 1.416 2 5.580 7 假交替单胞菌属 Pseudoalteromonas 0.210 7 0.391 1 6.549 9 弓形菌属 Arcobacter 0.567 3 1.984 1 4.513 1 假红杆菌属 Pseudorhodobacter 0.013 7 2.440 6 1.598 1 肉杆菌属 Carnobacterium 0.671 0 0.158 8 1.567 2
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