{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T04:30:10Z","timestamp":1786336210980,"version":"build-2736575974"},"reference-count":45,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T00:00:00Z","timestamp":1786147200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["23H02529"],"award-info":[{"award-number":["23H02529"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"award":["23H02529"],"award-info":[{"award-number":["23H02529"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004318","name":"Shimane University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100004318","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/01jaaym28","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Paramecium tritobursaria is a ciliate that harbors intracellular Chlorella sp. symbionts and serves as a model organism for studying endosymbiosis. After ingestion by P. tritobursaria, some algal cells are digested within digestive vacuoles (DVs), whereas others escape digestion and become enclosed by a perialgal vacuole (PV) membrane, establishing a stable symbiosis. We investigated whether inhibiting DV acidification with ammonium chloride (NH4Cl) modulates algal digestion and the establishment of endosymbiosis. Congo red-stained yeast assays showed that treatment with 20 mM NH4Cl for 40 min effectively suppressed DV acidification. Under these conditions, algal intracellular behavior was altered; escape from DVs appeared to be delayed, particularly at 6 h after uptake, and many cells remained within the DVs. Despite this delay, symbiosis was established at 24 h in both groups. Notably, NH4Cl treatment significantly increased the symbiosis establishment rate, with an average 1.5-fold increase and up to a 3-fold increase compared with the control. These results demonstrate that transient inhibition of DV acidification alters digestion dynamics and promotes the establishment of symbiosis. This study provides a novel strategy for dissecting P. tritobursaria\u2013Chlorella sp. endosymbiosis through controlled modulation of DV maturation.<\/jats:p>","DOI":"10.3390\/microorganisms14081742","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T01:20:03Z","timestamp":1786324803000},"page":"1742","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Effects of Ammonium Chloride-Mediated Control of Digestive Vacuole Acidification on Endosymbiosis Between Paramecium tritobursaria and Chlorella variabilis"],"prefix":"10.3390","volume":"14","author":[{"given":"Narumi","family":"Uchida","sequence":"first","affiliation":[{"name":"Agricultural and Life Sciences, Graduate School of Natural Science and Technology, Shimane University, Matsue-shi 690-8504, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1263-349X","authenticated-orcid":false,"given":"Yuuki","family":"Kodama","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Faculty of Life and Environmental Sciences, Shimane University, Matsue-shi 690-8504, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1111\/j.1550-7408.1974.tb03702.x","article-title":"Transfer of photosynthetically produced carbohydrate from endosymbiotic Chlorellae to Paramecium bursaria","volume":"21","author":"Brown","year":"1974","journal-title":"J. Protozool."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/BF00446890","article-title":"The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis. III. The influence of different CO2-concentrations and of glucose on the photosynthetic and respiratory capacity of the symbiotic unit","volume":"125","author":"Reisser","year":"1980","journal-title":"Arch. Microbiol."},{"key":"ref_3","first-page":"55","article-title":"Nitrogen nutrition of endosymbiotic Chlorella spec","volume":"1","author":"Albers","year":"1985","journal-title":"Endocytobiosis Cell Res."},{"key":"ref_4","first-page":"639","article-title":"Dissociation and restoration of endocellular symbiosis in Paramecium bursaria","volume":"134","author":"Siegel","year":"1959","journal-title":"Anat. Rec."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1111\/j.1558-5646.1965.tb01728.x","article-title":"Evolution and Symbiosis in the Genus Chlorella and Related Algae","volume":"19","author":"Karakashian","year":"1965","journal-title":"Evolution"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1111\/j.1749-6632.1987.tb40618.x","article-title":"Naturally Occurring and Artificially Established Associations of Ciliates and Algae","volume":"503","author":"Reisser","year":"1987","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Kodama, Y., Suzuki, H., Dohra, H., Sugii, M., Kitazume, T., Yamaguchi, K., Shigenobu, S., and Fujishima, M. (2014). Comparison of gene expression of Paramecium bursaria with and without Chlorella variabilis symbionts. BMC Genom., 15.","DOI":"10.1186\/1471-2164-15-183"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.1038\/s41598-017-01331-0","article-title":"Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics","volume":"7","author":"Song","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1360","DOI":"10.1038\/s41396-018-0341-4","article-title":"Genetic basis for the establishment of endosymbiosis in Paramecium","volume":"13","author":"He","year":"2019","journal-title":"ISME J."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Cheng, Y.-H., Liu, C.-F.J., Yu, Y.-H., Jhou, Y.-T., Fujishima, M., Tsai, I.J., and Leu, J.-Y. (2020). Genome plasticity in Paramecium bursaria revealed by population genomics. BMC Biol., 18.","DOI":"10.1186\/s12915-020-00912-2"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Jenkins, B.H. (2024). Mutualism on the edge: Understanding the Paramecium\u2013Chlorella symbiosis. PLoS Biol., 22.","DOI":"10.1371\/journal.pbio.3002563"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"293","DOI":"10.5507\/fot.2011.028","article-title":"Taxonomic reassessment of the genus Chlorella (Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species","volume":"11","author":"Bock","year":"2011","journal-title":"Fottea"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Piasecka, A., and Baier, A. (2022). Metabolic and Proteomic Analysis of Chlorella sorokiniana, Chloroidium saccharofilum, and Chlorella vulgaris Cells Cultured in Autotrophic, Photoheterotrophic, and Mixotrophic Cultivation Modes. Molecules, 27.","DOI":"10.3390\/molecules27154817"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S0932-4739(98)80023-0","article-title":"Species-specificity of Chlorella for establishment of symbiotic association with Paramecium bursaria\u2014Does infectivity depend upon sugar components of the cell wall?","volume":"34","author":"Takeda","year":"1998","journal-title":"Eur. J. Protistol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/s00709-006-0241-8","article-title":"Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process","volume":"231","author":"Kodama","year":"2007","journal-title":"Protoplasma"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"8216","DOI":"10.1038\/s41598-022-12496-8","article-title":"Endosymbiotic Chlorella variabilis reduces mitochondrial number in the ciliate Paramecium bursaria","volume":"12","author":"Kodama","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/s13068-016-0424-2","article-title":"Highly efficient lipid production in the green alga Parachlorella kessleri: Draft genome and transcriptome endorsed by whole-cell 3D ultrastructure","volume":"9","author":"Ota","year":"2016","journal-title":"Biotechnol. Biofuels"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1016\/j.apenergy.2012.07.031","article-title":"Recent advances in liquid biofuel production from algal feedstocks","volume":"102","author":"Daroch","year":"2013","journal-title":"Appl. Energy"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1038\/s41598-017-00979-y","article-title":"Development of New Carbon Resources: Production of Important Chemicals from Algal Residue","volume":"7","author":"Yamaguchi","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1007\/s10811-025-03708-5","article-title":"Experimental infection of aposymbiotic Paramecium bursaria with starch and oil-producing Parachlorella kessleri","volume":"38","author":"Bessyo","year":"2026","journal-title":"J. Appl. Phycol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s00709-005-0087-5","article-title":"Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection","volume":"225","author":"Kodama","year":"2005","journal-title":"Protoplasma"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Fujishima, M. (2009). Infection of Paramecium bursaria by Symbiotic Chlorella Species. Endosymbionts in Paramecium, Springer. Microbiology Monographs.","DOI":"10.1007\/978-3-540-92677-1"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Witzany, G., and Nowacki, M. (2016). Paramecium as a Model Organism for Studies on Primary and Secondary Endosymbioses. Biocommunication of Ciliates, Springer.","DOI":"10.1007\/978-3-319-32211-7"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Kodama, Y., and Fujishima, M. (2024). Effects of the Symbiotic Chlorella variabilis on the Host Ciliate Paramecium bursaria Phenotypes. Microorganisms, 12.","DOI":"10.3390\/microorganisms12122537"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1111\/1574-6941.12448","article-title":"Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria","volume":"90","author":"Kodama","year":"2014","journal-title":"FEMS Microbiol. Ecol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1007\/BF01322784","article-title":"Photosynthesis dependent acidification of perialgal vacuoles in the Paramecium bursaria\/Chlorella symbiosis: Visualization by monensin","volume":"166","author":"Schnepf","year":"1992","journal-title":"Protoplasma"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1111\/pre.12461","article-title":"Characteristics of maltose transport system in the endosymbiont Chlorella variabilis of Paramecium bursaria","volume":"69","author":"Shibata","year":"2021","journal-title":"Phycol. Res."},{"key":"ref_28","first-page":"412","article-title":"Phagosomal acidification in Paramecium: Effects on lysosomal fusion","volume":"43","author":"Fok","year":"1987","journal-title":"Eur. J. Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1111\/j.1550-7408.1984.tb02951.x","article-title":"Acid Phosphatase in the Digestive Vacuoles and Lysosomes of Paramecium caudatum: A Timed Study","volume":"31","author":"Fok","year":"1984","journal-title":"J. Protozool."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"18089","DOI":"10.1038\/s41598-022-22284-z","article-title":"Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts","volume":"12","author":"Spanner","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1111\/j.1550-7408.1966.tb01949.x","article-title":"The Syngens of Paramecium bursaria: New Mating Types and Intersyngenic Mating Reactions","volume":"13","author":"Bomford","year":"1966","journal-title":"J. Protozool."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1007\/s00709-010-0175-z","article-title":"Endosymbiosis of Chlorella species to the ciliate Paramecium bursaria alters the distribution of the host\u2019s trichocysts beneath the host cell cortex","volume":"248","author":"Kodama","year":"2011","journal-title":"Protoplasma"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1111\/j.1550-7408.1995.tb01550.x","article-title":"Evidence for a Viral Macronuclear Endosymbiont in Paramecium caudatum","volume":"42","author":"Tsukii","year":"1995","journal-title":"J. Eukaryot. Microbiol."},{"key":"ref_34","first-page":"s96","article-title":"Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation","volume":"6","author":"Dryl","year":"1959","journal-title":"J. Protozool."},{"key":"ref_35","first-page":"849","article-title":"Changes in Morphology, Buoyant Density and Protein Composition in Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa, a Macronucleus-Specific Symbiont of the Ciliate Paramecium caudatum","volume":"7","author":"Fujishima","year":"1990","journal-title":"Zool. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1084\/jem.174.4.881","article-title":"Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: Studies of a pathogenic mycobacterium and a nonpathogenic yeast","volume":"174","author":"Hart","year":"1991","journal-title":"J. Exp. Med."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Fujishima, M., and Kawai, M. (1997). Acidification in Digestive Vacuoles is an Early Event Required for Holospora Infection of Paramecium Nucleus. Eukaryotism and Symbiosis, Springer Nature.","DOI":"10.1007\/978-3-642-60885-8_28"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1111\/j.1550-7408.1982.tb05423.x","article-title":"The Correlation of Digestive Vacuole pH and Size with the Digestive Cycle in Paramecium caudatum","volume":"29","author":"Fok","year":"1982","journal-title":"J. Protozool."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.1128\/IAI.00586-16","article-title":"pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells","volume":"85","author":"Chua","year":"2017","journal-title":"Infect. Immun."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Westman, J., and Grinstein, S. (2021). Determinants of Phagosomal pH During Host-Pathogen Interactions. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.624958"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7039","DOI":"10.1128\/IAI.68.12.7039-7048.2000","article-title":"Phagosome acidification has opposite effects on intracellular survival of Bordetella pertussis and B. bronchiseptica","volume":"68","author":"Schneider","year":"2000","journal-title":"Infect. Immun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1640","DOI":"10.1172\/JCI119688","article-title":"Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation","volume":"100","author":"Levitz","year":"1997","journal-title":"J. Clin. Investig."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.ejop.2018.09.003","article-title":"Endosymbiosis-related changes in ultrastructure and chemical composition of Chlorella variabilis (Archaeplastida, Chlorophyta) cell wall in Paramecium bursaria (Ciliophora, Oligohymenophorea)","volume":"66","author":"Higuchi","year":"2018","journal-title":"Eur. J. Protistol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Xiong, J., Luu, T.T.T., Venkatachalam, K., Du, G., and Zhu, M.X. (2022). Glutamine Produces Ammonium to Tune Lysosomal pH and Regulate Lysosomal Function. Cells, 12.","DOI":"10.3390\/cells12010080"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.1128\/mBio.01187-17","article-title":"Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing","volume":"8","author":"Gilmartin","year":"2017","journal-title":"mBio"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1742\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T04:10:42Z","timestamp":1786335042000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1742"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,8]]},"references-count":45,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["microorganisms14081742"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14081742","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,8]]}}}