Morphological and Molecular Evidence of Microcystin-Producing Microcystis aeruginosa (Chroococcales, Cyanophyceae) from Pantabangan Lake, Nueva Ecija, Philippines
Keywords:
cyanobacterial harmful algal blooms, toxin biosynthesis genes, PCR detection, 16S rDNA phylogeny, water quality risk, bloom monitoringAbstract
Microcystin-producing cyanobacteria are widely known to be a significant threat to freshwater ecosystems, aquaculture, and public health. The principal aim of our study is to isolate, identify and detect Microcystis aeruginosa capable of producing microcystins from Pantabangan Lake, Philippines, using morphological characterisation and molecular approaches. Two strains of M. aeruginosa (GBJLPAN01 and GBTKPAN01) were established and their morphology and phylogeny were examined. Cells of M. aeruginosa were spherical, densely packed in irregular colonies and embedded within a mucilaginous sheath. Cell sizes ranged from 2 – 5 µm in diameter, and displayed a distinct blue-green colouration. Phylogenetic analyses inferred from 16S rDNA showed the clustering of our cultured strains within a well-supported Microcystis clade, though the genus exhibited non-monophyletic clustering. Additionally, phylogenetic analysis of mcyE gene sequences confirmed clustering with M. aeruginosa reference strains, providing molecular validation of species identification. PCR-based screening revealed the presence of mcyE gene in both laboratory cultures and field-collected water samples, indicating widespread potential for microcystin production in Pantabangan Lake. The presence of mcyE serves as a reliable molecular marker for toxigenic strains, enabling accurate assessment of microcystin biosynthetic capacity. This study provides the first verified molecular evidence of microcystin-producing M. aeruginosa in Pantabangan Lake, combining diagnostic morphological features with dual molecular confirmation (16S rDNA and mcyE phylogenies) including mcyE detection from both cultured isolates and field samples. Our findings highlight the need to recognise the potential risk of Microcystis blooms to aquatic life and water quality of the lake.
References
Baldia, S.F., Conaco, M.C.G., Nishijima, T., Imanishi, S. & Harada, K.I. (2003). Microcystin production during algal bloom occurrence in Laguna de Bay, the Philippines. Fisheries Science, 69, 110-116. DOI: 10.1046/j.1444-2906.2003.00594.x.
Bernardo, E.C., Mamauag, S.S. & Mendoza, J.B. (2020). Phytoplankton dynamics and trophic state in Paoay Lake, Northwestern Luzon, Philippines. Philippine Journal of Science, 149, 183–195.
Botes, D.P., Tuinman, A.A., Wessels, P.L., Viljoen, C.C., Kruger, H., Williams, D.H. & Hammond, S.J. (1984). The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Microcystis aeruginosa. Journal of the Chemical Society, Perkin Transactions, 1, 2311–2318. DOI: 10.1039/p19840002311.
Carmichael, W.W. (2001). Health effects of toxin-producing cyanobacteria: “The CyanoHABs.” Human and Ecological Risk Assessment: An International Journal, 7, 1393–1407. DOI: 10.1080/20018091095087.
Chorus, I. & Welker, M. (Eds.). (2021). Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management (2nd ed.): Taylor & Francis. pp. 858.
Conklin, K.Y., Stancheva, R., Otten, T.G., Fadness, R., Boyer, G.L., Read, B., Zhang, X. & Sheath, R.G. (2020). Molecular and morphological characterization of a novel dihydroanatoxin-a producing Microcoleus species (cyanobacteria) from the Russian River, California, USA. Harmful Algae, 93, 101767. DOI: 10.1016/j.hal.2020.101767.
Corbel, S., Mougin, C. & Bouaïcha, N. (2014). Cyanobacterial toxins: Modes of actions, fate in aquatic and soil ecosystems, phytotoxicity and bioaccumulation in agricultural crops. Chemosphere, 96, 1-15.
Cruz, R.V.O., Lasco, R.D., Pulhin, J.M., Pulhin, F.B. & Garcia, K.B. (2006). Climate change impact on water resources in Pantabangan Watershed, Philippines. In AIACC Final Technical Report. pp. 9-107.
Cuvin-Aralar, M.L.A., Nagasaki, K. & Onodera, H. (2002). Microcystins in natural blooms and laboratory cultured Microcystis aeruginosa from Laguna de Bay, Philippines. Fisheries Science, 68 (Suppl. 1), 152–155. DOI: 10.1078/0723-2020-00102.
Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. (2012). jModelTest 2: More models, new heuristics and parallel computing. Nature Methods, 9, 772. DOI: 10.1038/nmeth.2111.
Du, X., Liu, H., Yuan, L., Wang, Y., Ma, Y., Wang, R. & Zhang, H. (2019). The diversity of cyanobacterial toxins on structural characterization, distribution and identification: A systematic review. Toxins, 11, 530. DOI: 10.3390/toxins11090530.
Duan, X., Zhang, C., Struewing, I., Li, X., Allen, J. & Lu, J. (2022). Cyanotoxin-encoding genes as powerful predictors of cyanotoxin production during harmful cyanobacterial blooms in an inland freshwater lake: Evaluating a novel early-warning system. Science of the Total Environment, 830, 154568. DOI: 10.1016/j.scitotenv.2022.154568.
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology, 59, 307–321. DOI: 10.1093/sysbio/syq010.
Guiry, M.D. & Guiry, G.M. (2025). AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Retrieved from http://www.algae base.org
Haeder, D.P., Villafane, V.E. & Helbling, E.W. (2014). Productivity of aquatic primary producers under global climate change. Photochemical & Photobiological Sciences, 13, 1370-1392. DOI: 10.1039/C3PP50418B.
Hall, T.A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Humbert, J.F., Barbe, V., Latifi, A., Gugger, M., Calteau, A., Coursin, T. & de Marsac, N.T. (2013). A tribute to disorder in the genome of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. PLOS ONE, 8, e70747. DOI: 10.1371/journal.pone.0070747.
Ichimura, T. (1979). Media for the cultivation of algae. In T. Ichimura & Y. Tamiya (Eds.), Methods in Phycology. Tokyo: Japanese Society of Plant Physiologists. pp. 294–305.
Inog, Y.U.S.P., Racoma, C.J.M., Bate, J.M.B., Benico, G.A. & Tordesillas, D.T. (2025). Spatiotemporal variability of phytoplankton communities in the euphotic layers of a tropical caldera lake (Lake Taal, Batangas, Philippines). Biodiversitas Journal of Biological Diversity, 26, 3964–3973. DOI: 10.13057/biodiv/d260722.
Jensen, T., Saleh, L., Bents, D., Krohn, S., Wu, Y.C., Mucke, M. & Labes, A. (2023). Optimised protocols for RNA extraction from a broad taxonomic range of algae. Journal of Applied Phycology, 35, 1743-1753. DOI: 10.1007/s10811-023-02980-7.
Katoh, K. & Standley, D.M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780. DOI: 10.1093/molbev/mst010.
Komárek, J. & Anagnostidis, K. (1998). Cyanoprokaryota, Volume 1: Chroococcales. In H. Ettl, G. Gärtner, H. Heynig, & D. Mollenhauer (Eds.), Süßwasserflora von Mitteleuropa (Vol. 19/1). Gustav Fischer. pp. 1–548.
Komárek, J. & Komárková, J. (2002). Review of the European Microcystis-morphospecies (Cyanoprokaryotes) from nature. Czech Phycology, 2, 1-24.
Komárek, J., Komárková, J. & Kling, H. (2002). Filamentous cyanobacteria. In: Freshwater Algae of North America: Ecology and Classification. Academic Press. pp. 117–196.
Kumar, P., Rautela, A., Kesari, V., Szlag, D., Westrick, J. & Kumar, S. (2020). Recent developments in the methods of quantitative analysis of microcystins. Journal of Biochemical and Molecular Toxicology, 34, e22582. DOI: 10.1002/jbt.22582.
Moreira, C., Ramos, V., Azevedo, J. & Vasconcelos, V. (2014). Methods to detect cyanobacteria and their toxins in the environment. Applied Microbiology and Biotechnology, 98, 8073–8082. DOI: 10.1007/s00253-014-5951-9.
Neilan, B.A., Pearson, L.A., Muenchhoff, J., Moffitt, M.C. & Dittmann, E. (2013). Environmental conditions that influence toxin biosynthesis in cyanobacteria. Environmental Microbiology, 15, 1239-1253. DOI: 10.1111/j.1462-2920.2012.02729.x
Notario, E.F. (1964). A limnological study of Laguna de Bay with a special reference to the plankton (suspended organic matter). Unpublished manuscript, Freshwater Fisheries Division, Philippine Fisheries Commission, Manila: 10 p, 16 annexes. Retrieved from https://www.fao.org/4/ac0 09e/AC009E01.html
Nübel, U., Garcia-Pichel, F. & Muyzer, G. (1997). PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and environmental microbiology, 63 (8), 3327-3332. DOI: 10.1128/AEM.63.8.3327-3332.1997.
Nugumanova, G., Ponomarev, E.D., Askarova, S., Fasler-Kan, E. & Barteneva, N.S. (2023). Freshwater cyanobacterial toxins, cyanopeptides and neurodegenerative diseases. Toxins, 15, 233. DOI: 10.3390/toxins15030233.
Oberholster, P.J., Botha, A.M. & Grobbelaar, J.U. (2004). Microcystis aeruginosa: Source of toxic microcystins in drinking water. African Journal of Biotechnology, 3, 159–168. DOI: 10.5897/AJB2004.000-2029.
Olowe, B., Oluyege, J., Famurewa, O., Ogunniran, A. & Adelegan, O. (2017). Molecular identification of Escherichia coli and new emerging enteropathogen, Escherichia fergusonii, from drinking water sources in Ado-Ekiti, Ekiti State, Nigeria. Journal of Microbiology Research, 7, 45-54. DOI: 10.5923/j.microbiology.20170703.01
Otsuka, S., Suda, S., Li, R., Watanabe, M., Oyaizu, H., Matsumoto, S. & Watanabe, M.M. (1999). Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence. FEMS Microbiology Letters, 172, 15-21. DOI: 10.1111/ j.15746968.1999.tb13449.x
Otsuka, S., Suda, S., Li, R., Matsumoto, S. & Watanabe, M.M. (2001). Morphological variability of colonies of Microcystis morphospecies in culture. Journal of General and Applied Microbiology, 47, 61-70. DOI: 10.2323/jgam.46.39.
Papa, R.D.S. & Mamaril Sr, A.C. (2011). History of the biodiversity and limno-ecological studies on Lake Taal with notes on the current state of Philippine limnology. Philippine Science Letters, 4, 1-10.
Pulhin, J.M., Peras, R.J.J., Cruz, R.V.O., Lasco, R.D., Pulhin, F.B. & Tapia, M.A. (2006). Vulnerability of communities to climate variability and extremes: the Pantabangan-Carranglan watershed in the Philippines. AIACCWorking Paper. pp 44.
Rantala, A., Fewer, D.P., Hisbergues, M., Rouhiainen, L., Vaitomaa, J., Börner, T. & Sivonen, K. (2003). Phylogenetic evidence for the early evolution of microcystin synthesis. Proceedings of the National Academy of Sciences of the United States of America, 101, 568–573. DOI: 10.1073/pnas.03044 89101.
Rastogi, R.P., Madamwar, D. & Incharoensakdi, A. (2015). Bloom dynamics of cyanobacteria and their toxins: Environmental health impacts and mitigation strategies. Frontiers in Microbiology, 6, 1254. DOI: 10.3389/fmicb.2015.01254.
Rastogi, R.P., Sinha, R.P. & Incharoensakdi, A. (2014). The cyanotoxin-microcystins: current overview. Reviews in Environmental Science and Bio/Technology, 13, 215-249. DOI: 10.1007/s11157-014-9334-6.
Reyes, M.R. & Mendoza, V.B. (2019). The Pantabangan watershed management and erosion control project. In Forest and Watershed Development and Conservation in Asia and the Pacific. Routledge. pp. 485-556. DOI: 10.4324/ 9780429049583-8.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542. DOI: 10.1093/sysbio/sys029.
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiäinen, H., Buikema, W., Haselkorn, R. & Sivonen, K. (2000). Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90. Molecular Microbiology, 37, 156–167. DOI: 10.1046/j.1365-2958.2000.01977.x.
Szlag, D.C., Sinclair, J.L., Southwell, B. & Westrick, J.A. (2015). Cyanobacteria and cyanotoxins occurrence and removal from five high-risk conventional treatment drinking water plants. Toxins, 7, 2198-2220. DOI: 10.3390/toxins7062198.
Tabugo, S.R., Dalayap, R., Malaco, A., Alotaibi, A. & Cordero, M.A. (2023). High-throughput sequencing as a tool for detecting microbial communities in lake ecosystem and its implications in fish farming in Lake Buluan, Mindanao, Philippines. Polish Journal of Environmental Studies, 33, 391-404. DOI: 10.15244/pjoes/171935.
Tan, W., Liu, Y., Wu, Z., Lin, S., Yu, G., Yu, B. & Li, R. (2010). cpcBA-IGS as an effective marker to characterize Microcystis wesenbergii (Komárek) Komárek in Kondrateva (cyanobacteria). Harmful Algae, 9, 607-612. DOI: 10.1016/j.hal.2010.04.011.
Tanabe, Y., Kasai, F. & Watanabe, M.M. (2007). Multilocus sequence typing (MLST) reveals high genetic diversity and clonal population structure of the bloom-forming cyanobacterium Microcystis aeruginosa. Microbiology, 153, 3695–3703. DOI: 10.1099/mic.0.2007/010645-0.
Tayaban, K.M.M., Pintor, K.L. & Vital, P.G. (2018). Detection of potential harmful algal bloom-causing microalgae from freshwater prawn farms in Central Luzon, Philippines, for bloom monitoring and prediction. Environment, Development and Sustainability, 20, 1311-1328. DOI: 10.1007/s10668-017-9941-z.
Vaitomaa, J., Rantala, A., Halinen, K., Rouhiainen, L., Tallberg, P., Mokelke, L. & Sivonen, K. (2003). Quantitative real-time PCR for determination of microcystin synthetase E copy numbers for Microcystis and Anabaena in lakes. Applied and Environmental Microbiology, 69, 7289–7297. DOI: 10.1128/AEM.69.12.7289-7297.2003
Valério, E., Chambel, L., Paulino, S., Faria, N., Pereira, P. & Tenreiro, R. (2009). Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs. Microbiology, 155, 1887–1898. DOI: 10.1099/mic.0.026966-0.
Watanabe, M.F. & Oishi, S. (1980). Toxicities of Microcystis aeruginosa collected from some lakes, reservoirs, ponds and moat in Tokyo and adjacent regions. Japanese Journal of Limnology (Rikusuigaku Zasshi), 41, 5-9. DOI: 10.3739/rikusui.41.5.
Downloads
Additional Files
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Borneo Journal of Resource Science and Technology
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright Transfer Statement for Journal
1) In signing this statement, the author(s) grant UNIMAS Publisher an exclusive license to publish their original research papers. The author(s) also grant UNIMAS Publisher permission to reproduce, recreate, translate, extract or summarize, and to distribute and display in any forms, formats, and media. The author(s) can reuse their papers in their future printed work without first requiring permission from UNIMAS Publisher, provided that the author(s) acknowledge and reference publication in the Journal.
2) For open access articles, the author(s) agree that their articles published under UNIMAS Publisher are distributed under the terms of the CC-BY-NC-SA (Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License) which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes, provided the original work of the author(s) is properly cited.
3) For subscription articles, the author(s) agree that UNIMAS Publisher holds copyright, or an exclusive license to publish. Readers or users may view, download, print, and copy the content, for academic purposes, subject to the following conditions of use: (a) any reuse of materials is subject to permission from UNIMAS Publisher; (b) archived materials may only be used for academic research; (c) archived materials may not be used for commercial purposes, which include but not limited to monetary compensation by means of sale, resale, license, transfer of copyright, loan, etc.; and (d) archived materials may not be re-published in any part, either in print or online.
4) The author(s) is/are responsible to ensure his or her or their submitted work is original and does not infringe any existing copyright, trademark, patent, statutory right, or propriety right of others. Corresponding author(s) has (have) obtained permission from all co-authors prior to submission to the journal. Upon submission of the manuscript, the author(s) agree that no similar work has been or will be submitted or published elsewhere in any language. If submitted manuscript includes materials from others, the authors have obtained the permission from the copyright owners.
5) In signing this statement, the author(s) declare(s) that the researches in which they have conducted are in compliance with the current laws of the respective country and UNIMAS Journal Publication Ethics Policy. Any experimentation or research involving human or the use of animal samples must obtain approval from Human or Animal Ethics Committee in their respective institutions. The author(s) agree and understand that UNIMAS Publisher is not responsible for any compensational claims or failure caused by the author(s) in fulfilling the above-mentioned requirements. The author(s) must accept the responsibility for releasing their materials upon request by Chief Editor or UNIMAS Publisher.
6) The author(s) should have participated sufficiently in the work and ensured the appropriateness of the content of the article. The author(s) should also agree that he or she has no commercial attachments (e.g. patent or license arrangement, equity interest, consultancies, etc.) that might pose any conflict of interest with the submitted manuscript. The author(s) also agree to make any relevant materials and data available upon request by the editor or UNIMAS Publisher.
