弧菌噬菌体鸡尾酒制剂对凡纳滨对虾肠道微生物区系的影响

doi:10.3969/j.issn.1004-1524.20240886

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (5): 1045-1056.DOI: 10.3969/j.issn.1004-1524.20240886

弧菌噬菌体鸡尾酒制剂对凡纳滨对虾肠道微生物区系的影响

翁歆之¹(), 刁奕昕², 贺洁¹, 刘莉³, 沈海钰⁴, 郭琦³, 沈卫锋³, 韩明明³, 楼宝³, 吕孙建³^,^*()

1.台州市水产技术推广总站,浙江台州 310005
2.浙江万里学院生物与环境学院,浙江宁波 315100
3.浙江省农业科学院水生生物研究所,浙江杭州 310021
4.温岭市水产技术推广站,浙江温岭 317511

收稿日期:2024-10-17 出版日期:2025-05-25 发布日期:2025-06-11
作者简介:翁歆之(1987—),女,浙江台州人,硕士,研究方向为水产养殖和水生动物疫病防控。E-mail:wengxinzhi9@163.com
通讯作者: *吕孙建,E-mail:lvsunjian@163.com
基金资助:
浙江省产业团队项目“设施化养殖凡纳滨对虾生物安保体系构建与示范”;浙江省重点研发计划(2022C02027)

Effect of Vibrio phage cocktail on the intestinal microbiota of Litopenaeus vannamei

WENG Xinzhi¹(), DIAO Yixin², HE Jie¹, LIU Li³, SHEN Haiyu⁴, GUO Qi³, SHEN Weifeng³, HAN Mingming³, LOU Bao³, LYU Sunjian³^,^*()

1. Taizhou Municipal Aquatic Technology Extension Station, Taizhou 310005, Zhejiang, China
2. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
3. Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
4. Wenling Municipal Aquatic Technology Extension Station, Taizhou 317511, Zhejiang, China

Received:2024-10-17 Online:2025-05-25 Published:2025-06-11

摘要/Abstract

摘要：

本试验旨在研究噬菌体鸡尾酒制剂对凡纳滨对虾(Litopenaeus vannamei)肠道微生物区系的影响。选取帆布池养殖池塘,分离并鉴定环境中的弧菌,同时评估噬菌体鸡尾酒制剂对弧菌的抑菌效果。设置2组试验,每组3个养殖桶重复,噬菌体组饲喂含有噬菌体鸡尾酒制剂的饲料,对照组饲喂普通饲料,利用16S rRNA高通量测序技术测定肠道微生物区系差异。从帆布池分离到3株弧菌,分别命名为DXA(Vibrio. owensii)、DXD(V. harveyi)和DXF(Vibrio sp. VibC-Oc-076),噬菌体鸡尾酒制剂对3株弧菌抑菌效果较好。饲喂含有噬菌体鸡尾酒制剂的凡纳滨对虾肠道内微生物区系丰富度显著升高,且细菌菌群组成结构更稳定;门水平,对照组和噬菌体组优势菌群均为拟杆菌门、变形菌门和疣微菌门细菌,其中噬菌体组拟杆菌门没有明显变化;属水平,噬菌体组噬冷菌属和鲁杰氏菌属细菌丰度先上升后下降,鼠尾菌属细菌丰度先下降后上升。凡纳滨对虾肠道内弧菌属细菌丰度低于0.3%,饲用噬菌体鸡尾酒制剂的第2天弧菌数下降,但与对照组无显著差异。综上,弧菌噬菌体鸡尾酒制剂可以提高凡纳滨对虾肠道微生物区系多样性,而且能稳定肠道微生态环境,但对弧菌属细菌的影响与其本底丰度有一定关系,需要后续更多的研究来阐述其相关性。

关键词: 凡纳滨对虾, 弧菌, 噬菌体鸡尾酒制剂, 微生物区系

Abstract:

This experiment aimed to investigate the effects of a phage cocktail preparation on the intestinal microbiota of Litopenaeus vannamei. Vibrio strains were isolated and identified from canvas-pond aquaculture environments, while the antibacterial efficacy of the phage cocktail against Vibrio was evaluated. Two experimental groups were established, each with three replicate culture tanks. The phage group was fed with a diet containing the phage cocktail preparation, while the control group was fed with a standard diet. Differences in intestinal microbiota were analyzed using 16S rRNA high-throughput sequencing. Three Vibrio strains were isolated from the canvas ponds: DXA (Vibrio owensii), DXD (V. harveyi), and DXF (Vibrio sp. VibC-Oc-076), with the phage cocktail demonstrating effective inhibition against all three strains. Shrimp fed the phage cocktail exhibited significantly increased intestinal microbial richness and more stable bacterial community structures. At the phylum level, Bacteroidota, Proteobacteria, and Verrucomicrobiota dominated both groups, with no significant change observed in Bacteroidota abundance in the phage group. At the genus level, Algoriphagus and Ruegeria abundances initially increased then decreased in the phage group, while Muricauda abundance showed an opposite trend. The Vibrio genus abundance in shrimp intestines remained below 0.3%. Although Vibrio counts decreased on the second day of feeding with the phage cocktail, no significant difference was observed compared to the control group. In conclusion, the Vibrio-targeting phage cocktail enhanced the diversity of intestinal microbiota in L. vannamei and stabilized the gut microecological environment. However, its impact on Vibrio bacteria appeared related to baseline abundance levels, warranting further research to elucidate this correlation.

Key words: Litopenaeus vannamei, Vibrio, phage cocktail, microbiota

中图分类号:

S917.1

翁歆之, 刁奕昕, 贺洁, 刘莉, 沈海钰, 郭琦, 沈卫锋, 韩明明, 楼宝, 吕孙建. 弧菌噬菌体鸡尾酒制剂对凡纳滨对虾肠道微生物区系的影响[J]. 浙江农业学报, 2025, 37(5): 1045-1056.

WENG Xinzhi, DIAO Yixin, HE Jie, LIU Li, SHEN Haiyu, GUO Qi, SHEN Weifeng, HAN Mingming, LOU Bao, LYU Sunjian. Effect of Vibrio phage cocktail on the intestinal microbiota of Litopenaeus vannamei[J]. Acta Agriculturae Zhejiangensis, 2025, 37(5): 1045-1056.

图/表 11

图1 弧菌在TSA和TCBS培养基上的菌落形态

Fig.1 Colony morphology of Vibrio on TSA and TCBS media

图2 噬菌体的透射电子显微镜观察黄色箭头指示的为噬菌体。

Fig.2 Transmission electron microscopy observation of bacteriophages Bacteriophage indicated by yellow arrows.

图3 噬菌体效价测定 A、B和C分别为PVP48、PVP-XSN和PFNC-1噬菌体。

Fig.3 Determination of bacteriophage titers A, B, and C represent PVP48, PVP-XSN and PFNC-1 bacteriophages, respectively.

图4 滴注法测试噬菌体对3种弧菌的抑菌效果白色箭头指示的为滴加噬菌体鸡尾酒制剂的区域。

Fig.4 Spot test for evaluating the inhibitory effect of bacteriophages on three species of Vibrio White arrows indicate the regions where bacteriophage cocktail formulations were applied.

图5 凡纳滨对虾肠道微生物区系的维恩图 CK,对照组;EX,噬菌体组。

Fig.5 Venn diagram of intestinal microbiota of Litopenaeus vannamei CK, Control group; EX, Phage group.

图6 凡纳滨对虾肠道微生物区系α多样性的Chao1分析 (A)~(G)分别为第1~7天。标注不同字母代表具有显著性差异(P<0.05),标注相同字母代表无显著性差异(P>0.05)。下同。

Fig.6 Chao1 analysis of alpha diversity in intestinal microbiota of Litopenaeus vannamei (A)~(G) correspond to day 1 to day 7, respectively. Labeling different letters represents significant differences (P<0.05), and labeling the same letter represents no significant differences (P>0.05). The same as below.

图7 凡纳滨对虾肠道微生物区系α多样性的Shannon分析 (A)~(G)分别为第1~7天。

Fig.7 Shannon analysis of Alpha diversity in intestinal microbiota of Litopenaeus vannamei (A)~(G) correspond to day 1 to day 7, respectively.

图8 凡纳滨对虾肠道微生物区系的PCoA分析 (A)对照组;(B)噬菌体组。

Fig.8 PCoA analysis of intestinal microbiota of Litopenaeus vannamei (A) Control group; (B) Phage group.

图9 凡纳滨对虾肠道微生物区系的门水平分析 (A)对照组;(B)噬菌体组。

Fig.9 Phylum level analysis of intestinal microbiota of Litopenaeus vannamei (A) Control group; (B) Phage group.

图10 凡纳滨对虾肠道微生物区系差异的属水平分析 (A)对照组;(B)噬菌体组。

Fig.10 Genus level analysis of intestinal microbiota of Litopenaeus vannamei (A) Control group; (B) Phage group.

图11 凡纳滨对虾肠道弧菌的相对丰度

Fig.11 Relative abundance of intestinal Vibrio in Litopenaeus vannamei

参考文献 30

[1]	LI C Y, KUO H W, CHENG W. Enhancement of immunity and disease resistance in Litopenaeus vannamei through injection of tyramine formulated with polyethylene glycol[J]. Developmental & Comparative Immunology, 2024, 161: 105260.
[2]	周爽男. 涂塑帆布圆池循环水高密度养殖南美白对虾试验[J]. 水产养殖, 2024, 45(9): 44-45.
	ZHOU S N. Experiment on high-density culture of Penaeus vannamei with circulating water in plastic-coated canvas round pool[J]. Journal of Aquaculture, 2024, 45(9): 44-45. (in Chinese)
[3]	中华人民共和国农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2024中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2024.
[4]	DE LA PEÑA L D, MOQUERA G L, AMAR E C, et al. In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei[J]. Research in Veterinary Science, 2024, 171: 105204.
[5]	严必福. 南美白对虾病害防治案例[J]. 科学养鱼, 2003(10): 51.
	YAN B F. Case study on disease control of Penaeus vannamei[J]. Scientific Fish Farming, 2003(10): 51. (in Chinese)
[6]	WAIHO K, ABD RAZAK M S, ABDUL RAHMAN M Z, et al. A metagenomic comparison of clearwater, probiotic, and Rapid BFT^TM on Pacific whiteleg shrimp, Litopenaeus vannamei cultures[J]. PeerJ, 2023, 11: e15758.
[7]	FENG J M, HUANG Y X, HUANG M L, et al. A novel perlucin-like protein (PLP) protects Litopenaeus vannamei against Vibrio harveyi infection[J]. Fish & Shellfish Immunology, 2023, 139: 108932.
[8]	HOA T T T, FAGNON M S, THY D T M, et al. Growth performance and disease resistance against Vibrio parahaemolyticus of whiteleg shrimp (Litopenaeus vannamei) fed essential oil blend (Phyto AquaBiotic)[J]. Animals, 2023, 13(21): 3320.
[9]	BUNNOY A, YANGLANG A, TRIBAMRUNG N, et al. Dietary administration of yeast (Saccharomyces cerevisiae) hydrolysate from sugar byproducts promotes the growth, survival, immunity, microbial community and disease resistance to VP (AHPND) in Pacific white shrimp (Litopenaeus vannamei)[J]. Fish & Shellfish Immunology, 2024, 145: 109327.
[10]	李兵兵, 张茹, 刘靓, 等. 一起副溶血性弧菌食物中毒事件中菌株全基因组测序与耐药性分析[J]. 中国食品卫生杂志, 2024, 36(5): 528-532.
	LI B B, ZHANG R, LIU L, et al. Whole genome sequencing and antibiotic resistance analysis of Vibrio parahaemolyticus isolated from a food poisoning incidents[J]. Chinese Journal of Food Hygiene, 2024, 36(5): 528-532. (in Chinese with English abstract)
[11]	翟凯旋, 冷敦鹏, 李金香, 等. 噬菌体疗法: 治疗水产养殖领域细菌性疾病的绿色选择[J]. 山东畜牧兽医, 2024, 45(6): 96-99.
	ZHAI K X, LENG D P, LI J X, et al. Phage therapy: a green choice to treat bacterial diseases in aquaculture[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2024, 45(6): 96-99. (in Chinese)
[12]	KARUNASAGAR I, SHIVU M M, GIRISHA S K, et al. Biocontrol of pathogens in shrimp hatcheries using bacteriophages[J]. Aquaculture, 2007, 268(1/2/3/4): 288-292.
[13]	HAO Q F, BAI Y, ZHOU H L, et al. Isolation and characterization of bacteriophage VA5 against Vibrio alginolyticus[J]. Microorganisms, 2023, 11(12): 2822.
[14]	XU Y J, SUN J, HU J J, et al. Characterization and preliminary application of a novel lytic Vibrio parahaemolyticus bacteriophage vB_VpaP_SJSY21[J]. International Journal of Molecular Sciences, 2023, 24(24): 17529.
[15]	SINGHA B, RAWAT B S, VENKATARAMAN R, et al. Gut microbiome associated dysbiosis: limited regimens and expanding horizons of phage therapy[J]. Aspects of Molecular Medicine, 2023, 2: 100029.
[16]	FANG H H, ZHUANG Z X, HUANG L D, et al. A newly isolated strain of Haematococcus pluvialis GXU-A23 improves the growth performance, antioxidant and anti-inflammatory status, metabolic capacity and mid-intestine morphology of juvenile Litopenaeus vannamei[J]. Frontiers in Physiology, 2022, 13: 882091.
[17]	ANAS A, SUKUMARAN V, NAMPULLIPURACKAL DEVARAJAN D, et al. Probiotics inspired from natural ecosystem to inhibit the growth of Vibrio spp. causing white gut syndrome in Litopenaeus vannamei[J]. 3 Biotech, 2021, 11(2): 66.
[18]	LOMELÍ-ORTEGA C O, BARAJAS-SANDOVAL D R, MARTÍNEZ-VILLALOBOS J M, et al. A broad-host-range phage cocktail selectively and effectively eliminates Vibrio species from shrimp aquaculture environment[J]. Microbial Ecology, 2023, 86(2): 1443-1446.
[19]	THILLAICHIDAMBARAM M, NARAYANAN K, SELVARAJ S, et al. Isolation and characterization of Vibrio owensii from Palk Bay and its infection study against post larvae of Litopenaeus vannamei[J]. Microbial Pathogenesis, 2022, 172: 105751.
[20]	LIU H T, GUO S T, WANG R, et al. Pathogen of Vibrio harveyi infection and C-type lectin proteins in whiteleg shrimp (Litopenaeus vannamei)[J]. Fish & Shellfish Immunology, 2021, 119: 554-562.
[21]	STALIN N, SRINIVASAN P. Efficacy of potential phage cocktails against Vibrio harveyi and closely related Vibrio species isolated from shrimp aquaculture environment in the south east coast of India[J]. Veterinary Microbiology, 2017, 207: 83-96.
[22]	JUN J W, HAN J E, GIRI S S, et al. Phage application for the protection from acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannamei[J]. Indian Journal of Microbiology, 2018, 58(1): 114-117.
[23]	LOMELÍ-ORTEGA C O, MARTÍNEZ-DÍAZ S F. Phage therapy against Vibrio parahaemolyticus infection in the whiteleg shrimp (Litopenaeus vannamei) larvae[J]. Aquaculture, 2014, 434: 208-211.
[24]	吴立婷, 陆睿, 刘半红, 等. 产气荚膜梭菌噬菌体及乳酸杆菌联用改善肉鸡生长性能和调节肠道菌群功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2321-2332.
	WU L T, LU R, LIU B H, et al. Combination of Clostridium perfringens phage and Lactobacillus improves growth performance and regulates intestinal flora function of broilers[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(6): 2321-2332. (in Chinese with English abstract)
[25]	ZHAO H Z, LI Y, LV P L, et al. Salmonella phages affect the intestinal barrier in chicks by altering the composition of early intestinal flora: association with time of phage use[J]. Frontiers in Microbiology, 2022, 13: 947640.
[26]	YE J M, MENG Q, JIN K Z, et al. Phage cocktail alleviated type 2 diabetes by reshaping gut microbiota and decreasing proinflammatory cytokines[J]. Applied Microbiology and Biotechnology, 2023, 108(1): 9.
[27]	朱树娇, 张辉, 周艳, 等. 噬菌体复合制剂对肉鸡生长性能、免疫功能、肠道形态及肠道菌群的影响[J]. 动物营养学报, 2022, 34(5): 2928-2939.
	ZHU S J, ZHANG H, ZHOU Y, et al. Effects of phage cocktail preparation on growth performance, immune function, intestinal morphology and gut microbiota of broilers[J]. Chinese Journal of Animal Nutrition, 2022, 34(5): 2928-2939. (in Chinese with English abstract)
[28]	陈诗雨, 徐美余, 邓征宇, 等. 志贺氏菌噬菌体ΦDS8对患病肉鸡的治疗及其肠道菌群的影响[J]. 浙江农业学报, 2022, 34(7): 1386-1395.
	CHEN S Y, XU M Y, DENG Z Y, et al. Treatment of diseased broilers with Shigella bacteriophage ΦDS8 and the effect on their intestinal flora[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1386-1395. (in Chinese with English abstract)
[29]	AMOAH K, HUANG Q C, TAN B P, et al. Dietary supplementation of probiotic Bacillus coagulans ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response, and disease confrontation of Pacific white shrimp, Litopenaeus vannamei[J]. Fish & Shellfish Immunology, 2019, 87: 796-808.
[30]	SEVERIN G B, RAMLIDEN M S, FORD K C, et al. Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion[J]. mBio, 2023, 14(5): e0087523.

弧菌噬菌体鸡尾酒制剂对凡纳滨对虾肠道微生物区系的影响

Effect of Vibrio phage cocktail on the intestinal microbiota of Litopenaeus vannamei

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