{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T05:23:23Z","timestamp":1761801803853,"version":"build-2065373602"},"reference-count":43,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T00:00:00Z","timestamp":1761350400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32300096","32270114"],"award-info":[{"award-number":["32300096","32270114"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Accurately quantifying associated microbes is essential to understand the interactions between microplankton and their associated microbes. Most DNA-based methods, such as high-throughput sequencing, primarily assess the ratio of target objects to references in microplankton samples. However, simple random sampling (SRS) of individuals may lead to deviations in quantifying these ratios at the population level if these characteristics are associated with the reference content of individuals. This study considered group analysis, which involves detecting k groups with n individuals in each group, as an alternative approach and used simulated data based on the detection of Microcystis populations to evaluate the accuracy of different sampling plans. Our results indicate that increasing the number of individuals in each group could reduce sampling bias and improve the accuracy of comparisons between populations. Group analysis could also minimize the impact of the detection limit. This study demonstrated that, when detection methods only provide the ratio of target objects to references, group analysis is more appropriate than SRS for characterizing microplankton populations. Group analysis can be used not only for detecting associated microbes but also for identifying ingested organisms or the biochemical composition of microplankton. Our results also demonstrate how in situ individual-level studies support ecological investigations.<\/jats:p>","DOI":"10.3390\/biology14111493","type":"journal-article","created":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T02:50:48Z","timestamp":1761533448000},"page":"1493","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Sampling Method Considering Body Size for Detecting the Associated Microbes in Plankton Populations: A Case Study, Using the Bloom-Forming Cyanobacteria, Microcystis"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1969-3073","authenticated-orcid":false,"given":"Lizhou","family":"Lin","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China"},{"name":"Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"Guangdong Environmental Protection Key Laboratory of Microbiology and Regional Eco-Safety, Guangzhou 510070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nanqin","family":"Gan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Licheng","family":"Huang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"Kunming Dianchi and Plateau Lakes Institute, Kunming 650228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4300-2060","authenticated-orcid":false,"given":"Lirong","family":"Song","sequence":"additional","affiliation":[{"name":"Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4271-0134","authenticated-orcid":false,"given":"Liang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.hal.2015.12.005","article-title":"The interaction between cyanobacteria and zooplankton in a more eutrophic world","volume":"54","author":"Ger","year":"2016","journal-title":"Harmful Algae"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1111\/nph.15765","article-title":"Algae-bacteria interactions that balance the planktonic microbiome","volume":"223","author":"Cirri","year":"2019","journal-title":"New Phytol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"267","DOI":"10.5194\/bg-8-267-2011","article-title":"Distribution and host diversity of Amoebophryidae parasites across oligotrophic waters of the Mediterranean Sea","volume":"8","author":"Siano","year":"2011","journal-title":"Biogeosciences"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1111\/jpy.13132","article-title":"The Influence of Bacteria on the Growth, Lipid Production, and Extracellular Metabolite Accumulation by Phaeodactylum tricornutum (Bacillariophyceae)","volume":"57","author":"Chorazyczewski","year":"2021","journal-title":"J. Phycol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"101489","DOI":"10.1016\/j.algal.2019.101489","article-title":"Host selection and stochastic effects influence bacterial community assembly on the microalgal phycosphere","volume":"40","author":"Kimbrel","year":"2019","journal-title":"Algal Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.hal.2011.10.018","article-title":"Global toxicology, ecophysiology and population relationships of the chainforming PST dinoflagellate Gymnodinium catenatum","volume":"14","author":"Hallegraeff","year":"2012","journal-title":"Harmful Algae"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2225","DOI":"10.1021\/acs.chemrestox.0c00164","article-title":"Microcystins: Biogenesis, Toxicity, Analysis, and Control","volume":"33","author":"Schreidah","year":"2020","journal-title":"Chem. Res. Toxicol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Hu, L., Shan, K., Lin, L., Shen, W., Huang, L., Gan, N., and Song, L. (2016). Multi-Year Assessment of Toxic Genotypes and Microcystin Concentration in Northern Lake Taihu, China. Toxins, 8.","DOI":"10.3390\/toxins8010023"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2263","DOI":"10.1038\/s41396-018-0159-0","article-title":"Distinct patterns and processes of abundant and rare eukaryotic plankton communities following a reservoir cyanobacterial bloom","volume":"12","author":"Xue","year":"2018","journal-title":"ISME J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1038\/ismej.2011.113","article-title":"Coherent dynamics and association networks among lake bacterioplankton taxa","volume":"6","author":"Eiler","year":"2012","journal-title":"ISME J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1128\/aem.52.4.935-938.1986","article-title":"Effects of Flow Cytometric Analysis and Cell Sorting on Photosynthetic Carbon Uptake by Phytoplankton in Cultures and from Natural Populations","volume":"52","author":"Rivkin","year":"1986","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"532","DOI":"10.1016\/j.hal.2007.11.004","article-title":"Characterising the 2002 toxic Karenia concordia (Dinophyceae) outbreak and its development using satellite imagery on the north-eastern coast of New Zealand","volume":"7","author":"Chang","year":"2008","journal-title":"Harmful Algae"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.hal.2016.12.003","article-title":"A novel hematoxylin and eosin stain assay for detection of the parasitic dinoflagellate Amoebophrya","volume":"62","author":"Li","year":"2017","journal-title":"Harmful Algae"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.jembe.2018.09.006","article-title":"Does Noctiluca scintillans end the diatom bloom in coastal water?","volume":"510","author":"Kitatsuji","year":"2019","journal-title":"J. Exp. Mar. Biol. Ecol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.envpol.2007.12.027","article-title":"Non-microcystin producing Microcystis wesenbergii (Komarek) Komarek (Cyanobacteria) representing a main waterbloom-forming species in Chinese waters","volume":"156","author":"Xu","year":"2008","journal-title":"Environ. Pollut."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1080\/02705060.2010.9665068","article-title":"Community Structure of Bacteria Associated with Microcystis Colonies from Cyanobacterial Blooms","volume":"25","author":"Shia","year":"2010","journal-title":"J. Freshw. Ecol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1111\/1758-2229.12001","article-title":"Specific association between bacteria and buoyant Microcystis colonies compared with other bulk bacterial communities in the eutrophic Lake Taihu, China","volume":"4","author":"Shi","year":"2012","journal-title":"Environ. Microbiol. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1078\/0723202041748163","article-title":"Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: Detection of microcystins and microcystin genes in individual colonies","volume":"27","author":"Fastner","year":"2004","journal-title":"Syst. Appl. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1785","DOI":"10.1099\/mic.0.26947-0","article-title":"Diversity and distribution of Microcystis (Cyanobacteria) oligopeptide chemotypes from natural communities studied by single-colony mass spectrometry","volume":"150","author":"Welker","year":"2004","journal-title":"Microbiology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1016\/j.synbio.2023.11.001","article-title":"Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing","volume":"8","author":"Cui","year":"2023","journal-title":"Synth. Syst. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5659","DOI":"10.1016\/j.watres.2013.06.040","article-title":"Differences in microcystin production and genotype composition among Microcystis colonies of different sizes in Lake Taihu","volume":"47","author":"Wang","year":"2013","journal-title":"Water Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1016\/j.bse.2014.04.009","article-title":"Relationship between extracellular polysaccharides (EPS) content and colony size of Microcystis is colonial morphology dependent","volume":"55","author":"Zhu","year":"2014","journal-title":"Biochem. Syst. Ecol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1080\/02705060.2019.1665115","article-title":"Effects of the cultivable bacteria attached to Microcystis colonies on the colony size and growth of Microcystis","volume":"34","author":"Wu","year":"2019","journal-title":"J. Freshw. Ecol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1456","DOI":"10.1038\/ismej.2014.232","article-title":"Aerobic and anaerobic nitrogen transformation processes in N2-fixing cyanobacterial aggregates","volume":"9","author":"Klawonn","year":"2015","journal-title":"ISME J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5585","DOI":"10.1111\/mec.13844","article-title":"Metabarcoding and metabolome analyses of copepod grazing reveal feeding preference and linkage to metabolite classes in dynamic microbial plankton communities","volume":"25","author":"Ray","year":"2016","journal-title":"Mol. Ecol."},{"key":"ref_26","first-page":"1","article-title":"Review of the European Microcystis-morphospecies (Cyanoprokaryotes) from nature","volume":"2","year":"2002","journal-title":"Czech Phycol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"968","DOI":"10.2166\/wst.2022.277","article-title":"The role of attached bacteria in the formation of Microcystis colony in Chentaizi River","volume":"86","author":"Dai","year":"2022","journal-title":"Water Sci. Technol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4931","DOI":"10.1111\/1462-2920.16133","article-title":"Gene expression in the microbial consortia of colonial Microcystis aeruginosa-a potential buoyant particulate biofilm","volume":"24","author":"Shi","year":"2022","journal-title":"Environ. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Zhao, L., Lin, L.Z., Zeng, Y., Teng, W.K., Chen, M.Y., Brand, J.J., Zheng, L.L., Gan, N.Q., Gong, Y.H., and Li, X.Y. (2023). The facilitating role of phycospheric heterotrophic bacteria in cyanobacterial phosphonate availability and Microcystis bloom maintenance. Microbiome, 11.","DOI":"10.1186\/s40168-023-01582-2"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3020","DOI":"10.1111\/1462-2920.15514","article-title":"Individual Microcystis colonies harbour distinct bacterial communities that differ by Microcystis oligotype and with time","volume":"23","author":"Smith","year":"2021","journal-title":"Environ. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Perez-Carrascal, O.M., Tromas, N., Terrat, Y., Moreno, E., Giani, A., Marques, L.C.B., Fortin, N., and Shapiro, B.J. (2021). Single-colony sequencing reveals microbe-by-microbiome phylosymbiosis between the cyanobacterium Microcystis and its associated bacteria. Microbiome, 9.","DOI":"10.1186\/s40168-021-01140-8"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1051\/agro:19850504","article-title":"Group analysis using ELISA: Determination of the level of transmission of SOybean Mosaic Virus in soybean seed","volume":"5","author":"Maury","year":"1985","journal-title":"Agronomie"},{"key":"ref_33","first-page":"1132","article-title":"Progress in sampling inspection by Group Attributes","volume":"30","author":"Yang","year":"2011","journal-title":"J. Appl. Stat. Manag."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1079\/SSR200166","article-title":"Statistical considerations in seed purity testing for transgenic traits","volume":"11","author":"Remund","year":"2001","journal-title":"Seed Sci. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.chemolab.2004.07.005","article-title":"Minimum cost acceptance sampling plans for grain control, with application to GMO detection","volume":"75","author":"Kobilinsky","year":"2005","journal-title":"Chemom. Intell. Lab. Syst."},{"key":"ref_36","unstructured":"Sournia, A. (1978). How many cells to count?. Phytoplankton Manual, UNESCO Press."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Wilson, A.E., Kaul, R.B., and Sarnelle, O. (2010). Growth rate consequences of coloniality in a harmful phytoplankter. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0008679"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1093\/nar\/gks808","article-title":"Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies","volume":"41","author":"Klindworth","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e2584","DOI":"10.7717\/peerj.2584","article-title":"VSEARCH: A versatile open source tool for metagenomics","volume":"4","author":"Rognes","year":"2016","journal-title":"PeerJ"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"10","DOI":"10.14806\/ej.17.1.200","article-title":"CUTADAPT removes adapter sequences from high-throughput sequencing reads","volume":"17","author":"Martin","year":"2011","journal-title":"EMBnet. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nmeth.3869","article-title":"DADA2: High-resolution sample inference from Illumina amplicon data","volume":"13","author":"Callahan","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1111\/j.1442-9993.1993.tb00438.x","article-title":"Non-parametric multivariate analyses of changes in community structure","volume":"18","author":"Clarke","year":"1993","journal-title":"Aust. J. Ecol."},{"key":"ref_43","first-page":"32","article-title":"A new method for non-parametric multivariate analysis of variance","volume":"26","author":"Anderson","year":"2001","journal-title":"Austral Ecol."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/11\/1493\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T05:18:59Z","timestamp":1761801539000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/11\/1493"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,25]]},"references-count":43,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["biology14111493"],"URL":"https:\/\/doi.org\/10.3390\/biology14111493","relation":{},"ISSN":["2079-7737"],"issn-type":[{"type":"electronic","value":"2079-7737"}],"subject":[],"published":{"date-parts":[[2025,10,25]]}}}