Abstract
The morbidity and mortality peak of farmed Procambarus clarkii occurs around May every year, a phenomenon known as “Black May” disease (BMD). Increasing evidence shows that the intestinal flora is closely related to host health. We analyzed and compared the microbiota of healthy and BMD-affected P. clarkii intestines. The results show that there was no significant difference in bacterial α-diversity (richness P=0.59; evenness P=0.43; and diversity P=0.052) between the diseased group and the control group. Four dominant phyla in the intestines of crayfish in the control group, namely Tenericutes (30.86%), Bacteroidetes (29.99%), Firmicutes (22.23%), and Proteobacteria (15.23%), were identified. However, a striking shift in the microbial composition were found in the intestines of P. clarkii with BMD. Bacteroidetes was a dominant phylum in healthy P. clarkii, whereas the prevalence was low in diseased P. clarkii (1.87%). By contrast, the prevalence of Proteobacteria was significantly higher (P<0.05) in P. clarkii with BMD than in P. clarkii without BMD. Candidatus Bacilloplasma, Bacteroides, Vibrio, and Aeromonas showed significant differences (P<0.05) at the genus level. Tax4Fun function prediction indicated that the relative abundance of genes involved in energy metabolism in the intestinal flora of P. clarkii with BMD was significantly reduced (P<0.05). Therefore, BMD can change the composition of the intestinal microbiota of P. clarkii. This study contributes to the understanding of the relationship between intestinal flora and host especially in aquatic animals.
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Data Availability Statement
All raw data were submitted to the NCBI Sequence Read Archive (SRA) under accession number PRJNA692534. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Alloul A, Wille M, Lucenti P, Bossier P, Van Stappen G, Vlaeminck S E. 2021. Purple bacteria as added-value protein ingredient in shrimp feed: Penaeus vannamei growth performance, and tolerance against Vibrio and ammonia stress. Aquaculture, 530: 735788, https://doi.org/10.1016/j.aquaculture.2020.735788.
Berry D, Reinisch W. 2013. Intestinal microbiota: a source of novel biomarkers in inflammatory bowel diseases? Best Practice & Research Clinical Gastroenterology, 27(1): 47–58, https://doi.org/10.1016/j.bpg.2013.03.005.
Berry D, Schwab C, Milinovich G, Reichert J, Ben Mahfoudh K, Decker T, Engel M, Hai B, Hainzl E, Heider S, Kenner L, Müller M, Rauch I, Strobl B, Wagner M, Schleper C, Urich T, Loy A. 2012. Phylotype-level 16S rRNA analysis reveals new bacterial indicators of health state in acute murine colitis. The ISME Journal, 6(11): 2091–2106, https://doi.org/10.1038/ismej.2012.39.
Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F D, Costello E K, Fierer N, Pena A G, Goodrich J K Gordon J I, Huttley G A, Kelley S T, Knights D, Koenig J E, Ley R E, Lozupone C A, McDonald D, Muegge B D, Pirrung M, Reeder J, Sevinsky J R, Turnbaugh P J, Walters W A, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5): 335–336, https://doi.org/10.1038/nmeth.003.
Chen X H. 2019. The reasons for the formation of “Black May” and prevention and control measures in rice field shrimp farming. Fishery Guide to be Rich, 16: 52–54.
Dai W F, Yu W N, Zhang J J, Zhu J Y, Tao Z, Xiong J B. 2017. The gut eukaryotic microbiota influences the growth performance among cohabitating shrimp. Applied Microbiology and Biotechnology, 101(16): 6447–6457, https://doi.org/10.1007/s00253-017-8388-0.
Dong P S, Guo H P, Wang Y T, Wang R Y, Chen H P, Zhao Y J, Wang K, Zhang D M. 2021. Gastrointestinal microbiota imbalance is triggered by the enrichment of Vibrio in subadult Litopenaeusvannamei with acute hepatopancreatic necrosis disease. Aquaculture, 533: 736199, https://doi.org/10.1016/j.aquaculture.2020.736199.
El Asmar R, Fasano A, Bamford P, Berti I, Not T, Catassi C, Coppa G V, Panigrahi P. 2000. Probiotics prevent zonulin-mediated intestinal barrier dysfunction secondary to bacterial colonization. Gastroenterology, 118(4): A815.
Fan L F, Li Q X. 2019. Characteristics of intestinal microbiota in the Pacific white shrimp Litopenaeus vannamei differing growth performances in the marine cultured environment. Aquaculture, 505: 450–461, https://doi.org/10.1016/j.aquaculture.2019.02.075.
Gherardi F. 2006. Crayfish invading Europe: the case study of Procambarus clarkii. Marine and Freshwater Behaviour and Physiology, 39(3): 175–191, https://doi.org/10.1080/10236240600869702.
Gregory K E, LaPlante R D, Shan G, Kumar D V, Gregas M. 2015. Mode of birth influences preterm infant intestinal colonization with Bacteroides over the early neonatal period. Advances in Neonatal Care, 15(6): 386–393, https://doi.org/10.1097/ANC.0000000000000237.
Guarner F. 2007. Role of intestinal flora in health and disease. Nutrición Hospitalaria, 22(S2): 14–19.
Guo X, Xia X, Tang R, Zhou J, Zhao H, Wang K. 2008. Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Letters in Applied Microbiology, 47(5): 367–373, https://doi.org/10.1111/j.1472-765X.2008.02408.x.
Haas B J, Gevers D, Earl A M, Feldgarden M, Ward D V, Giannoukos G, Ciulla D, Tabbaa D, Highlander S K, Sodergren E, Methe B, DeSantis T Z, The Human Microbiome Consortium, Petrosino J F, Knight R, Birren B W. 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Research, 21(3): 494–504, https://doi.org/10.1101/gr.112730.110.
Hou D W, Huang Z J, Zeng S Z, Liu J, Wei D D, Deng X S, Weng S P, Yan Q Y, He J G. 2018. Intestinal bacterial signatures of white feces syndrome in shrimp. Applied Microbiology and Biotechnology, 102(8): 3701–3709, https://doi.org/10.1007/s00253-018-8855-2.
Huang P D, Shen G Q, Gong J, Zhu M R, Wang Y, Zhang X, Hashimu Ame K, Zang Y N, Shen H S. 2020. A novel Dicistro-like virus discovered in Procambarus clarkii with “Black May” disease. Journal of Fish Diseases, 44(6): 803–811, https://doi.org/10.1111/jfd.13309.
Huang Z B, Li X Y, Wang L P, Shao Z Z. 2016. Changes in the intestinal bacterial community during the growth of white shrimp, Litopenaeus vannamei. Aquaculture Research, 47(6): 1737–1746, https://doi.org/10.1111/are.12628.
Karlsson F H, Ussery D W, Nielsen J, Nookaew I. 2011. A closer look at Bacteroides: phylogenetic relationship and genomic implications of a life in the human gut. Microbial Ecology, 61(3): 473–485, https://doi.org/10.1007/s00248-010-9796-1.
Klase G, Lee S, Liang S, Kim J, Zo Y G, Lee J. 2019. The microbiome and antibiotic resistance in integrated fishfarm water: Implications of environmental public health. Science of the Total Environment, 649: 1491–1501, https://doi.org/10.1016/j.scitotenv.2018.08.288.
Kostanjsek R, Strus J, Avgustin G. 2007. “Candidatus Bacilloplasma,” a novel lineage of Mollicutes associated with the hindgut wall of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Applied and Environmental Microbiology, 73(17): 5566–5573, https://doi.org/10.1128/AEM.02468-06.
Li E C, Xu C, Wang X D, Wang S F, Zhao Q, Zhang M L, Qin J G, Chen L Q. 2018. Gut microbiota and its modulation for healthy farming of pacific white shrimp Litopenaeus vannamei. Reviews in Fisheries Science & Aquaculture, 26(3): 381–399, https://doi.org/10.1080/23308249.2018.1440530.
Li T T, Long M, Ji C, Shen Z X, Gatesoupe F J, Zhang X J, Zhang Q Q, Zhang L L, Zhao Y L, Liu X H, Li A H. 2016. Alterations of the gut microbiome of largemouth bronze gudgeon (Coreius guichenoti) suffering from furunculosis. Scientific Reports, 6(1): 30606, https://doi.org/10.1038/srep30606.
Li Y D, Zhou F L, Tang Y P, Huang J H, Yang L S, Jiang S, Yang Q B, Jiang S G. 2021. Variation in bacterial communities among stress-sensitive and stress-tolerant black tiger shrimp (Penaeus monodon) individuals. Aquaculture Research, 52(5): 2146–2159, https://doi.org/10.1111/are.15067.
Liu Q, Long Y N, Li B, Zhao L L, Luo J, Xu L, Luo W, Du Z J, Zhou J, Yang S. 2020a. Rice-shrimp culture: a better intestinal microbiota, immune enzymatic activities, and muscle relish of crayfish (Procambarus clarkii) in Sichuan Province. Applied Microbiology and Biotechnology, 104(21): 9413–9420, https://doi.org/10.1007/s00253-020-10797-4.
Liu X D, He X, An Z H, Sun W, Chen N, Gao X J, Li X X, Zhang X J. 2020b. Citrobacter freundii infection in red swamp crayfish (Procambarus clarkii) and host immune-related gene expression profiles. Aquaculture, 515: 734499, https://doi.org/10.1016/j.aquaculture.2019.734499.
Magoč T, Salzberg S L. 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 27(21): 2957–2963, https://doi.org/10.1093/bioinformatics/btr507.
Othman M, Agüero R, Lin H C. 2008. Alterations in intestinal microbial flora and human disease. Current Opinion in Gastroenterology, 24(1): 11–16, https://doi.org/10.1097/MOG.0b013e3282f2b0d7.
Ott S J, Musfeldt M, Wenderoth D F, Hampe J, Brant O, Fölsch U R, Timmis K N, Schreiber S. 2004. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut, 53(5): 685–693, https://doi.org/10.1136/gut.2003.025403.
Rognes T, Flouri T, Nichols B, Quince C, Mahé F. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ, 4: e2584, https://doi.org/10.7717/peerj.2584.
Sekirov I, Russell S L, Antunes L C M, Finlay B B. 2010. Gut microbiota in health and disease. Physiological Reviews, 90(3): 859–904, https://doi.org/10.1152/physrev.00045.2009.
Semova I, Carten J D, Stombaugh J, Mackey L C, Knight R, Farber S A, Rawls J F. 2012. Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish. Cell Host & Microbe, 12(3): 277–288, https://doi.org/10.1016/j.chom.2012.08.003.
Serrano-Villar S, Rojo D, Martinez-Martinez, M, Deusch S, Vazquez-Castellanos J F, Bargiela R, Sainz T, Vera M, Moreno S, Estrada V, Jose Gosalbes M, Latorre A, Seifert J, Barbas C, Moya A, Ferrer M, 2016. Gut bacteria metabolism impacts immune recovery in HIV-infected individuals. Ebiomedicine, 8: 203–216, https://doi.org/10.1016/j.ebiom.2016.04.033.
Shen G Q, Zhang X, Gong J, Wang Y, Huang P D, Shui Y, Xu Z H, Shen H S. 2020. Transcriptomic analysis of Procambarus clarkii affected by “Black May” disease. Scientific Reports, 10(1): 21225, https://doi.org/10.1038/s41598-020-78191-8.
Shi H N, Walker A. 2004. Bacterial colonization and the development of intestinal defences. Canadian Journal of Gastroenterology and Hepatology, 18: 690421, https://doi.org/10.1155/2004/690421.
Shui Y, Guan Z B, Liu G F, Fan L M. 2020. Gut microbiota of red swamp crayfish Procambarus clarkii in integrated crayfish-rice cultivation model. AMB Express, 10(1): 5, https://doi.org/10.1186/s13568-019-0944-9.
Wang W, Wen B H, Gasparich G E, Zhu N N, Rong L W, Chen J X, Xu Z K. 2004. A spiroplasma associated with tremor disease in the Chinese mitten crab (Eriocheir sinensis). Microbiology, 150(9): 3035–3040, https://doi.org/10.1099/mic.0.26664-0.
Watanabe M, Houten S M, Mataki C, Christoffolete M A, Kim B W, Sato H, Messaddeq N, Harney J W, Ezaki O, Kodama T, Schoonjans K, Bianco A C, Auwerx J. 2006. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature, 439(7075): 484–489, https://doi.org/10.1038/nature04330.
Xiong J B, Wang K, Wu J F, Qiuqian L L, Yang K J, Qian Y X, Zhang D M. 2015. Changes in intestinal bacterial communities are closely associated with shrimp disease severity. Applied Microbiology and Biotechnology, 99(16): 6911–6919, https://doi.org/10.1007/s00253-015-6632-z.
Xu J, Mahowald M A, Ley R E, Lozupone C A, Hamady M, Martens E C, Henrissat B, Coutinho P M, Minx P, Latreille P, Cordum H, Van Brunt A, Kim K, Fulton R S, Fulton L A, Clifton S W, Wilson R K, Knight R D, Gordon J I. 2007. Evolution of symbiotic bacteria in the distal human intestine. PLoS Biology, 5(7): e156, https://doi.org/10.1371/journal.pbio.0050156.
Yue G H, Wang G L, Zhu B Q, Wang C M, Zhu Z Y, Lo L C. 2008. Discovery of four natural clones in a crayfish species Procambarus clarkii. International Journal of Biological Sciences, 4(5): 279–282.
Zeng T L, Ye Y F, Mu C K, Wang K, Li R H, Wang C L. 2016. Gut Microbiota and metabolic phenotype of Portunus trituberculatus. Chinese Journal of Analytical Chemistry, 44(12): 1867–1873, https://doi.org/10.1016/S1872-2040(16)60978-7.
Zhang Y, Li Z Y, Kholodkevich S, Sharov A, Chen C, Feng Y J, Ren N Q, Sun K. 2020. Effects of cadmium on intestinal histology and microbiota in freshwater crayfish (Procambarus clarkii). Chemosphere, 242: 125105, https://doi.org/10.1016/j.chemosphere.2019.125105.
Zhang Y, Sun K, Li Z Y, Chai X X, Fu X Y, Kholodkevich S, Kuznetsova T, Chen C, Ren N Q. 2021. Effescts of acute diclofenac exposure on intestinal histology, antioxidant defense, and microbiota in freshwater crayfish (Procambarus clarkii). Chemosphere, 263: 128130, https://doi.org/10.1016/j.chemosphere.2020.128130.
Zhong Y D, Marungruang N, Fak F, Nyman M. 2015. Effects of two whole-grain barley varieties on caecal SCFA, gut microbiota and plasma inflammatory markers in rats consuming low- and high-fat diets. British Journal of Nutrition, 113(10): 1558–1570, https://doi.org/10.1017/S0007114515000793.
Zhou Y T, Zhi F C. 2016. Lower level of Bacteroides in the gut microbiota is associated with inflammatory bowel disease: a meta-analysis. Biomed Research International, 2016: 5828959, https://doi.org/10.1155/2016/5828959.
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Supported by the Natural Science Fund Project of Jiangsu Province (No. BK20181138) and the Central Public-Interest Scientific Institution Basal Research Fund, Freshwater Fisheries Research Center, CAFS (No. 2019JBFZ09)
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Gong, J., Shen, G., Zhu, M. et al. 16S rRNA gene sequencing analysis on changes in the intestinal flora of Procambarus clarkii with “Black May” disease. J. Ocean. Limnol. 40, 2068–2079 (2022). https://doi.org/10.1007/s00343-021-1278-4
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DOI: https://doi.org/10.1007/s00343-021-1278-4