循环水养殖系统中凡纳滨对虾肠道微生物对三种复合益生菌制剂的响应
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摘要
【目的】在循环养殖系统中应用不同的复合益生菌制剂,探讨凡纳滨对虾肠道菌群结构特征及免疫水平发生的变化。【方法】30 d养殖周期结束后,通过平板计数法分析肠道细菌总数和弧菌总数;基于高通量测序技术分析肠道样品V3+V4区菌群特征;采用实时荧光定量PCR技术分析免疫相关因子TLR1和Dorsal基因表达水平,阐述益生菌制剂应用的意义。【结果】益生菌制剂的应用降低了凡纳滨对虾肠道中细菌总数,抑制弧菌的生长,间接预防疾病的发生。不同益生菌制剂从不同程度上优化了凡纳滨对虾肠道菌群结构,提高高质量序列和有效OTU数量,Chao1、Simpson、Shannon指数显示了丰富度和多样性变化,复合益生菌制剂3效果较好。同时,菌群结构得到优化,其中益生菌制剂1组对拟杆菌门含量百分比产生显著影响。Toll受体TLR1和Toll通路中的Dorsal基因m RNA表达受到益生菌制剂的影响,1、3组促进了TLR 1表达,1、2、3组都促进了Dorsal基因表达。【结论】在循环水养殖系统中添加益生菌制剂可优化凡纳滨对虾肠道菌群特征,提高免疫水平,为病害防控和健康养殖提供理论依据。
[Objective] We studied the effects of three biological agents on intestinal microbiota and immunity of white shrimp(Litopenaeus vannamei) in a water-recirculating cultivation system.[Methods] The composition of three biological agents were Bacillus subtilis,Candida utilis,nitrifying bacteria and photosynthetic bacteria(treatment 1);Proteinase,Acidophilus spp.,Bacillus subtilis and Faecalis spp.(treatment 2);and Protease,yeast,Bacillus subtilis and Lactobacillus acidophilus(treatment 3).After feeding 30 days,the total numbers of bacteria and Vibrio in the intestine of L.vannamei were calculated using plate counting.The high throughput sequencing of 16 S r DNA gene's V3+V4 region was used to determine diversity and composition of bacterial communities.Real time fluorescence quantitative PCR was used to analyze the expression levels of Toll-like receptor 1(TLR1) and Dorsal.[Results] After the application of biological agents,the total number of bacteria in the intestine of L.vannamei was reduced to 7.22 CFU/g,and the growth of Vibrio was inhibited(5.01 CFU/g) in treatment 3.In treatment 3,the percentage of high sequences and Operational Taxonomic Unit(OTU) were increased to 98.8% and 326,Chao1,Simpson,Shannon was 581.34,0.94,5.52 respectively,which displayed the changes of bacterium abundance and diversity in intestine.Furthermore,the community structure of intestinal microbiota was optimized.Proteobacteria,Bacteroidetes,Actinobacteria,Verrucomicrobia,Firmicutes,Candidatus Saccharibacteria and Cyanobacteria were the main dominant phyla.However,the proportion of Bacteroidetes was significantly increased(40.7%) in the treatment 1(P<0.05).The different expression levels of Toll-like receptor 1 and Dorsal were changed in different probiotics.In treatment 3,the expression level of Toll-like receptor 1(TLR1) was 3.2(P<0.05),which was 220% higher than control,meanwhile,the expression level of Dorsal was 4.7(P<0.05),which was 370% higher than control.[Conclusion] Our study revealed that biological agents played an active role in the recirculating system,which could optimize the structure of intestinal microbiota and improve the expression levels of immune genes.In this case,these biological agents could serve as candidates for diseases prevention and healthy aquaculture.And the treatment 3 was the better.
[Objective] We studied the effects of three biological agents on intestinal microbiota and immunity of white shrimp(Litopenaeus vannamei) in a water-recirculating cultivation system.[Methods] The composition of three biological agents were Bacillus subtilis,Candida utilis,nitrifying bacteria and photosynthetic bacteria(treatment 1);Proteinase,Acidophilus spp.,Bacillus subtilis and Faecalis spp.(treatment 2);and Protease,yeast,Bacillus subtilis and Lactobacillus acidophilus(treatment 3).After feeding 30 days,the total numbers of bacteria and Vibrio in the intestine of L.vannamei were calculated using plate counting.The high throughput sequencing of 16 S r DNA gene's V3+V4 region was used to determine diversity and composition of bacterial communities.Real time fluorescence quantitative PCR was used to analyze the expression levels of Toll-like receptor 1(TLR1) and Dorsal.[Results] After the application of biological agents,the total number of bacteria in the intestine of L.vannamei was reduced to 7.22 CFU/g,and the growth of Vibrio was inhibited(5.01 CFU/g) in treatment 3.In treatment 3,the percentage of high sequences and Operational Taxonomic Unit(OTU) were increased to 98.8% and 326,Chao1,Simpson,Shannon was 581.34,0.94,5.52 respectively,which displayed the changes of bacterium abundance and diversity in intestine.Furthermore,the community structure of intestinal microbiota was optimized.Proteobacteria,Bacteroidetes,Actinobacteria,Verrucomicrobia,Firmicutes,Candidatus Saccharibacteria and Cyanobacteria were the main dominant phyla.However,the proportion of Bacteroidetes was significantly increased(40.7%) in the treatment 1(P<0.05).The different expression levels of Toll-like receptor 1 and Dorsal were changed in different probiotics.In treatment 3,the expression level of Toll-like receptor 1(TLR1) was 3.2(P<0.05),which was 220% higher than control,meanwhile,the expression level of Dorsal was 4.7(P<0.05),which was 370% higher than control.[Conclusion] Our study revealed that biological agents played an active role in the recirculating system,which could optimize the structure of intestinal microbiota and improve the expression levels of immune genes.In this case,these biological agents could serve as candidates for diseases prevention and healthy aquaculture.And the treatment 3 was the better.
引文
[1]Zheng XT,Duan YF,Dong HB,Yang K,Yang YY,Zhang JS.Effects of Lactobacillus plantarum on growth performance,gut histology and activity of digestive enzymes in Pacific White Leg Shrimp,Litopenaeus vannamei.Fisheries Science,2016,35(1):1–6.(in Chinese)郑晓婷,段亚飞,董宏标,杨铿,杨莺莺,张家松.植物乳酸杆菌对凡纳滨对虾生长、消化酶活性和肠道组织形态的影响.水产科学,2016,35(1):1–6.
[2]Wang YX,Wang GX,Zhang W.Application technology of photosynthetic bacteria and Bacillus in the aquaculture of White Prawn.Fisheries of Heilongjiang,2008,(2):22–23.(in Chinese)王越新,王桂香,张巍.光合细菌和芽孢杆菌在南美白对虾养殖中的应用技术.黑龙江水产,2008,(2):22–23.
[3]Rengpipat S,Rukpratanporn S,Piyatiratitivorakul S,et al.Immunity enhancement in black tiger shrimp(Penaeus monodon)by a probiont bacterium(Bacillus S11).Aquaculture,2000,191(4):271–288.
[4]Gullian M,Thompson F,Rodriguez J.Selection of probiotic bacteria and study of their immunostimulatory effect in Penaeus vannamei.Aquaculture,2004,233(1/4):1–14.
[5]Yuan H,Wang JK.KBMR mieroalgae shrimp brood stock and clam seeds integrated recirculating production system.//AES Session:Microalgae-based Recirculation Systems Recirculating aquaculture Production Systems.World Aquaculture,Bali,Indonesia,2005.
[6]Li CH,Wei XL,Wang XF,Jie XY,Hu WA,Lu QY,Liu L,Xu JJ.Technical characteristics and trend of cultivation pattern under the recycling water in shrimp pond.Guangdong Agricultural Sciences,2011,37(24):105–108.(in Chinese)李纯厚,魏小岚,王学锋,颉晓勇,胡维安,陆琴燕,刘璐,徐娇娇.池塘循环水对虾养殖模式的技术特点及发展趋势.广东农业科学,2011,37(24):105–108.
[7]Drengstig A,Bergheim A.Commercial land-based farming of European lobster(Homarus gammarus L.)in recirculating aquaculture system(RAS)using a single cage approach.Aquacultural Engineering,2013,53:14–18.
[8]Tan J,Luo K,Luan S,Cao BX,Guo ZJ,Xu SY,Kong J.Preliminary application of the recirculating aquaculture system in Litopenaeus Vannamei breeding.Periodical of Ocean University of China,2016,6(4):63–70.(in Chinese)谭建,罗坤,栾生,曹宝祥,郭兆加,许圣钰,孔杰.循环水养殖系统在凡纳滨对虾种虾养殖中的应用效果初探.中国海洋大学学报,2016,6(4):63–70.
[9]Chen GZ,Zhang BL,Ji MM,Wu XG,Zhou JY,Chen JN,Wang Y,Tian H,Zhang XJ.Gut microbiota of polystyrene-eating mealworms analyzed by high-throughput sequencing.Microbiology China,2017,44(6):2011–2018.(in Chinese)陈冠舟,张白鹭,纪梦梦,吴晓刚,周君仪,陈家楠,王芸,田浩,张晓君.高通量测序探究啮食聚苯乙烯泡沫塑料黄粉虫的肠道菌群结构.微生物学通报,2017,44(6):2011–2018.
[10]Guo TF,Huang XX,Su M,Wang WG,Huai XJ,Hu P,Yan JQ,Lv D,Huang ZZ.Effects of zinc supplementation in diet on the immunity,Vibrio-resistant ability,lysozyme m RNA and Toll receptor m RNA expressions in the white shrimp(Litopenaeus vannamei).Journal of Fisheries of China,2011,35(7):1081–1089.(in Chinese)郭腾飞,黄旭雄,苏明,王武刚,怀向军,胡盼,严佳琦,吕丹,黄征征.饲料锌添加水平对凡纳滨对虾免疫抗菌机能和溶菌酶m RNA及Toll受体m RNA表达的影响.水产学报,2011,35(7):1081–1089.
[11]Qi CC,Wang BJ,Liu M,Jiang KY,Zhao W,Ren CH,Hu CQ,Wang L.Effects of short term addition of aflatoxin B1(AFB1)on the intestinal mucosal barrier of Litopenaeus vannamei.Journal of Fisheries of China,2017,41(12):1936–1945.(in Chinese)齐灿灿,王宝杰,刘梅,蒋克勇,赵伟,任春华,胡超群,王雷.黄曲霉毒素B1(AFB1)的短期投喂对凡纳滨对虾肠道黏膜屏障的影响.水产学报,2017,41(12):1936–1945.
[12]Hu Y,Tan BP,Mai KS,Ai QH,Zheng SX,Cheng KM.Effects of dietary probiotic on growth,immunity and intestinal bacteria of juvenile Litopenaeus vannamei.Journal of Fishery Sciences of China,2008,15(2):244–251.(in Chinese)胡毅,谭北平,麦康森,艾庆辉,郑石轩,程开敏.饲料中益生菌对凡纳滨对虾生长、肠道菌群及部分免疫指标的影响.中国水产科学,2008,15(2):244–251.
[13]Vaseeharan B,Ramasamy P.Control of pathogenic Vibrio spp.by Bacillus subtilis BT23,a possible probiotic treatment for black tiger shrimp Penaeus monodon.Letters in Applied Microbiology,2003,36:83–87.
[14]Tannock GW.Studies of the intestinal microflora:a prerequisite for the development of probiotics.International Dairy Journal,1998,8(5/6):527–533.
[15]李玉宏.养殖对虾肠道微生物区系的特征与功能.上海海洋大学硕士学位论文,2013.
[16]Wang XH,Li HR,Zhang XH,Li Y,Ji WS,Xu HS.Microbial flora in the digestive tract of adult penaeid shrimp(Penaeus chinensis).Journal of Ocean University of Qingdao,2000,30(3):493–498.
[17]Li JQ,Tan BP,Mai KS.Dietary probiotic Bacillus OJ and isomaltooligosaccharides influence the intestine microbial populations,immune responses and resistance to white spot syndrome virus in shrimp(Litopenaeus vannamei).Aquaculture,2009,291(1/2):35–40.
[18]Zhou ZG,Ding ZK,Huiyuan LV.Effects of dietary short-chain fructooligosaccharides on intestinal microflora,survival,and growth performance of juvenile white shrimp,Litopenaeus vannamei.Journal of the World Aquaculture Society,2007,38(2):296–301.
[19]Rendueles O,Ferrières L,Frétaud M,Bégaud E,Herbomel P,Levraud JP,Ghigo JM.A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria.PLo S Pathogens,2012,8(7):e1002815.
[20]Wang TF,Jin FJ,Ma SY,Chang ZY,Zhao YL,Gao HL.Research on microbial diversity in aquicultural water of Penaeus vannamei with DGGE technique.Biotechnology Bulletin,2012,(10):131–136.(in Chinese)王亭芳,金风杰,马士禹,常忠义,赵云龙,高红亮.DGGE技术对南美白对虾养殖水体中微生物多样性的研究.生物技术通报,2012,(10):131–136.
[21]Xue M,He YY,Qiu MD,Liang HF,Chen DX,Wang LQ,Liu YF,Wen CQ.Characterization of aquatic bacterial community of Litopenaeus vannamei larvae during hatchery period with high-throughput sequencing.Journal of Fisheries of China,2017,41(5):785–794.(in Chinese)薛明,何瑶瑶,邱孟德,梁华芳,陈第省,王立群,刘杨锋,温崇庆.高通量测序分析凡纳滨对虾育苗期水体菌群结构特征.水产学报,2017,41(5):785–794.
[22]Tang Y,Liu WL,Song XL,Zhang XJ,Zhang SJ,Huang J.Effects of dietary with Bacillus cereus on the growth rate and intestinal microflora of Litopenaeus vannamei.Journal of Fisheries of China,2017,41(5):765–774.(in Chinese)唐杨,刘文亮,宋晓玲,张晓静,张盛静,黄倢.饲料中补充蜡样芽孢杆菌对凡纳滨对虾生长及其肠道微生物组成的影响.水产学报,2017,41(5):765–774.
[23]Zhang ML,Sun YH,Chen K,Yu N,Zhou ZG,Chen LQ,Du ZY,Li EC.Characterization of the intestinal microbiota in Pacific white shrimp,Litopenaeus vannamei,fed diets with different lipid sources.Aquaculture,2014,434:449–455.
[24]Rungrassamee W,Klanchui A,Maibunkaew S,Chaiyapechara S,Jiravanichpaisal P,Karoonuthaisiri N.Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp(Penaeus monodon).PLo S One,2014,9(3):e91853.
[25]Ligoxygakis P,Pelte N,Hoffmann JA,Reichhart JM.Activation of Drosophila toll during fungal infection by a blood serine protease.Science,2002,297(5578):114–116.
[26]Hoffmann JA.The immune response of Drosophila.Nature,2003,426(6962):33–38.
[27]Imler JL,Hoffmann JA.Toll receptors in innate immunity.Trends in Cell Biology,2001,11(7):304–311.
[28]Feng W,Li J,Li JT,Chen P,Liao MJ.Effects of supplemental different level Vc on survival and non-specific immunity of Chinese shrimp(Fenneropenaeus chinensis).Journal of Fisheries of China,2011,35(2):200–207.(in Chinese)冯伟,李健,李吉涛,陈萍,廖梅杰.Vc对中国对虾非特异免疫因子及TLR/NF-κB表达量的影响.水产学报,2011,35(2):200–207.
[2]Wang YX,Wang GX,Zhang W.Application technology of photosynthetic bacteria and Bacillus in the aquaculture of White Prawn.Fisheries of Heilongjiang,2008,(2):22–23.(in Chinese)王越新,王桂香,张巍.光合细菌和芽孢杆菌在南美白对虾养殖中的应用技术.黑龙江水产,2008,(2):22–23.
[3]Rengpipat S,Rukpratanporn S,Piyatiratitivorakul S,et al.Immunity enhancement in black tiger shrimp(Penaeus monodon)by a probiont bacterium(Bacillus S11).Aquaculture,2000,191(4):271–288.
[4]Gullian M,Thompson F,Rodriguez J.Selection of probiotic bacteria and study of their immunostimulatory effect in Penaeus vannamei.Aquaculture,2004,233(1/4):1–14.
[5]Yuan H,Wang JK.KBMR mieroalgae shrimp brood stock and clam seeds integrated recirculating production system.//AES Session:Microalgae-based Recirculation Systems Recirculating aquaculture Production Systems.World Aquaculture,Bali,Indonesia,2005.
[6]Li CH,Wei XL,Wang XF,Jie XY,Hu WA,Lu QY,Liu L,Xu JJ.Technical characteristics and trend of cultivation pattern under the recycling water in shrimp pond.Guangdong Agricultural Sciences,2011,37(24):105–108.(in Chinese)李纯厚,魏小岚,王学锋,颉晓勇,胡维安,陆琴燕,刘璐,徐娇娇.池塘循环水对虾养殖模式的技术特点及发展趋势.广东农业科学,2011,37(24):105–108.
[7]Drengstig A,Bergheim A.Commercial land-based farming of European lobster(Homarus gammarus L.)in recirculating aquaculture system(RAS)using a single cage approach.Aquacultural Engineering,2013,53:14–18.
[8]Tan J,Luo K,Luan S,Cao BX,Guo ZJ,Xu SY,Kong J.Preliminary application of the recirculating aquaculture system in Litopenaeus Vannamei breeding.Periodical of Ocean University of China,2016,6(4):63–70.(in Chinese)谭建,罗坤,栾生,曹宝祥,郭兆加,许圣钰,孔杰.循环水养殖系统在凡纳滨对虾种虾养殖中的应用效果初探.中国海洋大学学报,2016,6(4):63–70.
[9]Chen GZ,Zhang BL,Ji MM,Wu XG,Zhou JY,Chen JN,Wang Y,Tian H,Zhang XJ.Gut microbiota of polystyrene-eating mealworms analyzed by high-throughput sequencing.Microbiology China,2017,44(6):2011–2018.(in Chinese)陈冠舟,张白鹭,纪梦梦,吴晓刚,周君仪,陈家楠,王芸,田浩,张晓君.高通量测序探究啮食聚苯乙烯泡沫塑料黄粉虫的肠道菌群结构.微生物学通报,2017,44(6):2011–2018.
[10]Guo TF,Huang XX,Su M,Wang WG,Huai XJ,Hu P,Yan JQ,Lv D,Huang ZZ.Effects of zinc supplementation in diet on the immunity,Vibrio-resistant ability,lysozyme m RNA and Toll receptor m RNA expressions in the white shrimp(Litopenaeus vannamei).Journal of Fisheries of China,2011,35(7):1081–1089.(in Chinese)郭腾飞,黄旭雄,苏明,王武刚,怀向军,胡盼,严佳琦,吕丹,黄征征.饲料锌添加水平对凡纳滨对虾免疫抗菌机能和溶菌酶m RNA及Toll受体m RNA表达的影响.水产学报,2011,35(7):1081–1089.
[11]Qi CC,Wang BJ,Liu M,Jiang KY,Zhao W,Ren CH,Hu CQ,Wang L.Effects of short term addition of aflatoxin B1(AFB1)on the intestinal mucosal barrier of Litopenaeus vannamei.Journal of Fisheries of China,2017,41(12):1936–1945.(in Chinese)齐灿灿,王宝杰,刘梅,蒋克勇,赵伟,任春华,胡超群,王雷.黄曲霉毒素B1(AFB1)的短期投喂对凡纳滨对虾肠道黏膜屏障的影响.水产学报,2017,41(12):1936–1945.
[12]Hu Y,Tan BP,Mai KS,Ai QH,Zheng SX,Cheng KM.Effects of dietary probiotic on growth,immunity and intestinal bacteria of juvenile Litopenaeus vannamei.Journal of Fishery Sciences of China,2008,15(2):244–251.(in Chinese)胡毅,谭北平,麦康森,艾庆辉,郑石轩,程开敏.饲料中益生菌对凡纳滨对虾生长、肠道菌群及部分免疫指标的影响.中国水产科学,2008,15(2):244–251.
[13]Vaseeharan B,Ramasamy P.Control of pathogenic Vibrio spp.by Bacillus subtilis BT23,a possible probiotic treatment for black tiger shrimp Penaeus monodon.Letters in Applied Microbiology,2003,36:83–87.
[14]Tannock GW.Studies of the intestinal microflora:a prerequisite for the development of probiotics.International Dairy Journal,1998,8(5/6):527–533.
[15]李玉宏.养殖对虾肠道微生物区系的特征与功能.上海海洋大学硕士学位论文,2013.
[16]Wang XH,Li HR,Zhang XH,Li Y,Ji WS,Xu HS.Microbial flora in the digestive tract of adult penaeid shrimp(Penaeus chinensis).Journal of Ocean University of Qingdao,2000,30(3):493–498.
[17]Li JQ,Tan BP,Mai KS.Dietary probiotic Bacillus OJ and isomaltooligosaccharides influence the intestine microbial populations,immune responses and resistance to white spot syndrome virus in shrimp(Litopenaeus vannamei).Aquaculture,2009,291(1/2):35–40.
[18]Zhou ZG,Ding ZK,Huiyuan LV.Effects of dietary short-chain fructooligosaccharides on intestinal microflora,survival,and growth performance of juvenile white shrimp,Litopenaeus vannamei.Journal of the World Aquaculture Society,2007,38(2):296–301.
[19]Rendueles O,Ferrières L,Frétaud M,Bégaud E,Herbomel P,Levraud JP,Ghigo JM.A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria.PLo S Pathogens,2012,8(7):e1002815.
[20]Wang TF,Jin FJ,Ma SY,Chang ZY,Zhao YL,Gao HL.Research on microbial diversity in aquicultural water of Penaeus vannamei with DGGE technique.Biotechnology Bulletin,2012,(10):131–136.(in Chinese)王亭芳,金风杰,马士禹,常忠义,赵云龙,高红亮.DGGE技术对南美白对虾养殖水体中微生物多样性的研究.生物技术通报,2012,(10):131–136.
[21]Xue M,He YY,Qiu MD,Liang HF,Chen DX,Wang LQ,Liu YF,Wen CQ.Characterization of aquatic bacterial community of Litopenaeus vannamei larvae during hatchery period with high-throughput sequencing.Journal of Fisheries of China,2017,41(5):785–794.(in Chinese)薛明,何瑶瑶,邱孟德,梁华芳,陈第省,王立群,刘杨锋,温崇庆.高通量测序分析凡纳滨对虾育苗期水体菌群结构特征.水产学报,2017,41(5):785–794.
[22]Tang Y,Liu WL,Song XL,Zhang XJ,Zhang SJ,Huang J.Effects of dietary with Bacillus cereus on the growth rate and intestinal microflora of Litopenaeus vannamei.Journal of Fisheries of China,2017,41(5):765–774.(in Chinese)唐杨,刘文亮,宋晓玲,张晓静,张盛静,黄倢.饲料中补充蜡样芽孢杆菌对凡纳滨对虾生长及其肠道微生物组成的影响.水产学报,2017,41(5):765–774.
[23]Zhang ML,Sun YH,Chen K,Yu N,Zhou ZG,Chen LQ,Du ZY,Li EC.Characterization of the intestinal microbiota in Pacific white shrimp,Litopenaeus vannamei,fed diets with different lipid sources.Aquaculture,2014,434:449–455.
[24]Rungrassamee W,Klanchui A,Maibunkaew S,Chaiyapechara S,Jiravanichpaisal P,Karoonuthaisiri N.Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp(Penaeus monodon).PLo S One,2014,9(3):e91853.
[25]Ligoxygakis P,Pelte N,Hoffmann JA,Reichhart JM.Activation of Drosophila toll during fungal infection by a blood serine protease.Science,2002,297(5578):114–116.
[26]Hoffmann JA.The immune response of Drosophila.Nature,2003,426(6962):33–38.
[27]Imler JL,Hoffmann JA.Toll receptors in innate immunity.Trends in Cell Biology,2001,11(7):304–311.
[28]Feng W,Li J,Li JT,Chen P,Liao MJ.Effects of supplemental different level Vc on survival and non-specific immunity of Chinese shrimp(Fenneropenaeus chinensis).Journal of Fisheries of China,2011,35(2):200–207.(in Chinese)冯伟,李健,李吉涛,陈萍,廖梅杰.Vc对中国对虾非特异免疫因子及TLR/NF-κB表达量的影响.水产学报,2011,35(2):200–207.