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饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响
刘文亮,许 华,唐 杨,宋晓玲,黄 倢
作者单位
刘文亮 上海海洋大学 上海 201306农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
许 华 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
唐 杨 上海海洋大学 上海 201306农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
宋晓玲 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
黄 倢 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071 
摘要:
为了研究外源补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及对其肠道微生物组成的影响,以基础饲料为空白组,在基础饲料中添加蜡样芽孢杆菌游离态活菌作为游离态组(活菌含量为108 CFU/g);在基础饲料中添加蜡样芽孢杆菌活菌生物膜作为生物膜组(活菌含量为108 CFU/g);每组8个平行。在养殖大棚暂养7 d后,对凡纳滨对虾进行40 d的养殖实验。在第17天进行白斑综合征(White Spot Syndrome Virus,WSSV)攻毒实验;第22天进行副溶血弧菌(Vibrio parahaemolyticus)攻毒实验。期间在第1、5、10、15天采样,称重并测量体长计算生长速率。在第1、5、10、15、20、25天采样,并取其肠道内容物提取DNA,用16S rDNA序列V3+V4区高通量测序方法检测对虾肠道内微生物群落的结构及变化情况。结果显示,生物膜组和游离态组对虾体重、体长增长速率高于空白组,生物膜组与游离态组对虾体重、体长差异性不显著,生物膜组和游离态组对虾体重、体长与空白组相比差异性显著。实验中凡纳滨对虾肠道微生物由变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)等组成,其中,变形菌门平均占到总量的94.0%。变形菌门中主要以弧菌属(Vibrio)、发光杆菌属(Photobacterium)、Octadecabacter等为主,生物膜组、游离态组、空白对照组弧菌属平均含量分别为34.65%、39.27%、58.00%。在WSSV攻毒实验中,生物膜组、游离态组、空白对照组平均累积死亡率分别为80.0%、77.0%、92.0%,各组差异性不显著(P>0.05)。在副溶血弧菌攻毒实验中,生物膜组、游离态组、空白对照组平均累积死亡率分别为61.3%、75.0%、77.3%,生物膜组与游离态组和空白对照组相比差异性显著(P<0.05)。研究表明,饲料中添加蜡样芽孢杆菌生物膜和游离态蜡样芽孢杆菌投喂凡纳滨对虾后,可改变对虾肠道的微生物组成,提高凡纳滨对虾生长速度、增强抗病能力,而且添加蜡样芽孢杆菌生物膜效果最明显。
关键词:  蜡样芽孢杆菌  生物膜  凡纳滨对虾  肠道微生物  高通量测序
DOI:10.11758/yykxjz.20160314001
分类号:
基金项目:
The Effect of Diet with Bacillus cereus Biofilm on the Growth Rate, Disease Resistance and Intestinal Microflora of Litopenaeus vannamei
LIU Wenliang,XU Hua,TANG Yang,SONG Xiaoling,HUANG Jie
Abstract:
Immune response of Litopenaeus vannamei fed with biofilm and planktonic cells of Bacillus cereus was studied by evaluating the growth rate, disease resistance and intestinal microflora. The biofilm and planktonic cells of B.cereus (PC465) were added to original feed as biofilm feed and planktonic feed respectively. Shrimps were maintained in 24 buckets with similar lighting and position in three groups. Biofilm group and planktonic group were fed with the concentration of 108 CFU/g of either biofilm or planktonic cells added feed for 15 d. Shrimps were challenged with white spot syndrome virus (WSSV) after 17 days post feeding in both experimental diet groups. Then a reverse gavage challenge with Vibrio parahaemolyticus has been tested on day 22. The growth rate of shrimp was evaluated on day 1, 5, 10, 15. The diversity and composition of bacterial communities of shrimp intestine were assessed by high throughput sequencing of 16S rDNA on day 1, 5, 10, 15, 20 and 25. The results showed that the growth rate and final weight of the biofilm group and the planktonic group were higher than the control group. The intestinal microflora of L.vannamei mainly consisted of Proteobacteria, while Bacteroidetes, Actinobacteria, Firmicutes, Verrucomicrobia, Cyanophyta, with the content of Proteobacteria accounting for about 94%. The proportion of Firmicutes in the shrimp intestine of the biofilm group (0.45%) and planktonic group (0.93%) was higher than the control group (0.02%). Proteobacteria mainly included Vibrio, Photobacterium, Pseudoalteromonas, etc. The proportion of Vibrio in the biofilm group, planktonic group and control group was 34.65%, 39.27%, and 58.00%, respectively. The cumulative mortalities of the biofilm group, planktonic group and control group challenged with WSSV were 80%, 77% and 92%, respectively. The cumulative mortalities of the biofilm group, planktonic group and control group challenged with V. parahaemolyticus were 61.3%, 75% and 77.3%, respectively. It is concluded that diet supplemented with B. cereus can change the intestinal microflora of L.vannamei, improve the disease resistance and enhance growth. The biofilm fed shrimps were more resistant to virus Vibrio and pathogens compared to that with planktonic fed and the control shrimps.
Key words:  Bacillus cereus  Biofilm  Litopenaeus vannamei  Intestinal microflora  High throughput sequencing