Kuznetsov A. V., Vainer V. I., Volkova Yu. M., Tsygankova V. M., Bochko D. N., Mukhanov V. S. Trichoplax sp. H2 cultivation and regeneration from body fragments and dissociated cell aggregates: Outlook for genetic modification. Marine Biological Journal, 2022, vol. 7, no. 3, pp. 60-79. URL: https://marine-biology.ru/mbj/article/view/353



Trichoplax sp. H2, a simple multicellular animal cultivated in the laboratory, was studied with the aim of its further genetic modification. The idea here is to introduce genetic information into a cell suspension after dissociation of the Trichoplax body into single cells, followed by their aggregation and regeneration of the resulting agglomerates into a viable animal. 1. We analyzed the dynamics of the Trichoplax growth in Petri dishes on Tetraselmis marina algal mats. Specimens were uniform on the exponential growth stage. 2. Trichoplaxes were cut radially in a post-traumatic regeneration research, and the regeneration of the obtained parts was investigated under a microscope. Growth and reproduction rate of animals on nutrient mats were determined that decreased as the animals had been cut. The missing part of the Trichoplax body was replaced by remodeling of remaining cells. 3. The animals after a vital staining were dissociated into single cells in a medium with no divalent cations. Pear-shaped or rounded cells were identified, as well as epithelial cells with flagella maintaining motion activity for more than 12 hours. 4. Trichoplax plates were disintegrated in the presence of 10 μM amlodipine to quantify a cell population using flow cytometry. As estimated, Trichoplax (0.5–1 mm in size) consists of approximately 10,000 cells. 5. Treatment of animals with 10 % BSA (Bovine Serum Albumin) during various exposure intervals suggests a hypothesis on the existence of totipotent cells at the periphery of the Trichoplax body, probably in the rim. 6. In the course of reparative regeneration experiments, we achieved Trichoplax dissociation into single cells with 0.1 % BSA treatment and the following recreation of the viable organisms by centrifugation of a cell suspension and subsequent dispersion of a large pellet into fragments up to 0.1 mm prior to plating multicellular aggregates on nutrient mats. 7. The development of the aggregates was accompanied by active motion of cells and epithelialization of the surface, which resulted in cell growth, formation of a plate, and further vegetative division of Trichoplax. As assumed, the artificial stage of a single cell in a line of asexual reproductions allows to introduce foreign genetic information into Trichoplax, for example, in order to study the signal processing, organization, and functioning of this multicellular organism. Transgenesis, which is based on the dissociation of an animal body into single cells, could be applied to other organisms with high regenerative potential.


A. V. Kuznetsov

leading researcher, D. Sc.



V. I. Vainer
Yu. M. Volkova
V. M. Tsygankova
D. N. Bochko
V. S. Mukhanov

leading researcher, PhD




Кузнецов А. В., Кулешова О. Н., Пронозин А. Ю., Кривенко О. В., Завьялова О. С. Действие прямоугольных электрических импульсов низкой частоты на трихоплакса (тип Placozoa) // Морской биологический журнал. 2020a. Т. 5, № 2. С. 50–66. [Kuznetsov A. V., Kuleshova O. N., Pronozin A. Yu., Krivenko O. V., Zavyalova O. S. Effects of low frequency rectangular electric pulses on Trichoplax (Placozoa). Morskoj biologicheskij zhurnal, 2020a, vol. 5, no. 2, pp. 50–66. (in Russ.)]. https://doi.org/10.21072/mbj.2020.05.2.05

Романова Д. Ю. Разнообразие клеточных типов у гаплотипа H4 Placozoa sp. // Морской биологический журнал. 2019. Т. 4, № 1. С. 81–90. [Romanova D. Y. Cell types diversity of H4 haplotype Placozoa sp. Morskoj biologicheskij zhurnal, 2019, vol. 4, no. 1, pp. 81–90. (in Russ.)]. https://doi.org/10.21072/mbj.2019.04.1.07

Серавин Л. Н., Гудков А. В. Trichoplax adhaerens (тип Placozoa) – одно из самых примитивных многоклеточных животных. Санкт-Петербург : ТЕССА, 2005. 69 с. [Seravin L. N., Gudkov A. V. Trichoplax adhaerens (Placozoa) — odno iz samykh primitivnykh mnogokletochnykh zhivotnykh. Saint Petersburg : TESSA, 2005, 69 p. (in Russ.)]

Albertini M. C., Fraternale D., Semprucci F., Cecchini S., Colomba M., Rocchi M. B. L., Sisti D., Di Giacomo B., Mari M., Sabatini L., Cesaroni L., Balsamo M., Guidi L. Bioeffects of Prunus spinosa L. fruit ethanol extract on reproduction and phenotypic plasticity of Trichoplax adhaerens Schulze, 1883 (Placozoa). PeerJ, 2019, vol. 7, art. no. e6789 (22 p.). https://doi.org/10.7717/peerj.6789

Armon S., Bull M. S., Aranda-Diaz A., Prakash M. Ultrafast epithelial contractions provide insights into contraction speed limits and tissue integrity. Proceedings of the National Academy of Sciences, 2018, vol. 115, no. 44, pp. E10333–E10341. https://doi.org/10.1073/pnas.1802934115

Bond C. Continuous cell movements rearrange anatomical structures in intact sponge. Journal of Experimental Zoology, 1992, vol. 263, iss. 3, pp. 284–302. https://doi.org/10.1002/jez.1402630308

Currie J. D., Kawaguchi A., Traspas R. M., Schuez M., Chara O., Tanaka E. M. Live imaging of axolotl digit regeneration reveals spatiotemporal choreography of diverse connective tissue progenitor pools. Developmental Cell, 2016, vol. 39, iss. 4, pp. 411–423. https://doi.org/10.1016/j.devcel.2016.10.013

Dellaporta S. L., Xu A., Sagasser S., Jakob W., Moreno M. A., Buss L. W., Schierwater B. Mitochondrial genome of Trichoplax adhaerens supports Placozoa as the basal lower metazoan phylum. Proceedings of the National Academy of Sciences, 2006, vol. 103, no. 23, pp. 8751–8756. https://doi.org/10.1073/pnas.0602076103

DuBuc T. Q., Ryan J. F., Martindale M. Q. “Dorsal–ventral” genes are part of an ancient axial patterning system: Evidence from Trichoplax adhaerens (Placozoa). Molecular Biology and Evolution, 2019, vol. 6, iss. 5, pp. 966–973. https://doi.org/10.1093/molbev/msz025

Eitel M., Guidi L., Hadrys H., Balsamo M., Schierwater B. New insights into placozoan sexual reproduction and development. PLoS One, 2011, vol. 6, iss. 5, art. no. e19639 (9 p.). https://doi.org/10.1371/journal.pone.0019639

Eitel M., Osigus H. J., DeSalle R., Schierwater B. Global diversity of the Placozoa. PLoS One, 2013, vol. 8, iss. 4, art. no. e57131 (12 p.). https://doi.org/10.1371/journal.pone.0057131

Eitel M., Schierwater B. The phylogeography of the Placozoa suggests a taxon-rich phylum in tropical and subtropical waters. Molecular Ecology, 2010, vol. 19, iss. 11, pp. 2315–2327. https://doi.org/10.1111/j.1365-294X.2010.04617.x

Elkhatib W., Smith C. L., Senatore A. A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa. Journal of Biological Chemistry, 2019, vol. 294, iss. 44, pp. 16320–16336. https://doi.org/10.1074/jbc.RA119.010542

Galtsoff P. S. Regeneration after dissociation (an experimental study on sponges). II. Histogenesis of Microciona prolifera, verr. Journal of Experimental Zoology, 1925, vol. 42, iss. 1, pp. 223–255. https://doi.org/10.1002/jez.1400420110

Gildor T., Malik A., Sher N., Avraham L., Ben-Tabou de-Leon S. Quantitative developmental transcriptomes of the Mediterranean Sea urchin Paracentrotus lividus. Marine Genomics, 2016, vol. 25, pp. 89–94. https://doi.org/10.1016/j.margen.2015.11.013

Grell K. G. Eibildung und Furchung von Trichoplax adhaerens F. E. Schulze (Placozoa). Zeitschrift für Morphologie der Tiere, 1972, vol. 73, iss. 4, pp. 297–314. https://doi.org/10.1007/BF00391925

Grell K. G. Embryonalentwicklung bei Trichoplax adhaerens F. E. Schulze. Naturwissenschaften, 1971, vol. 58, iss. 11, pp. 570. https://doi.org/10.1007/BF00598728

Grell K. G., Benwitz G. Elektronenmikroskopische Beobachtungen über das Wachstum der Eizelle und die Bildung der „Befruchtungsmembran” von Trichoplax adhaerens F. E. Schulze (Placozoa). Zeitschrift für Morphologie der Tiere, 1974, vol. 79, iss. 4, pp. 295–310. https://doi.org/10.1007/BF00277511

Grell K. G., Benwitz G. Ergänzende Untersuchungen zur Ultrastruktur von Trichoplax adhaerens F. E. Schulze (Placozoa). Zoomorphology, 1981, vol. 98, iss. 1, pp. 47–67. https://doi.org/10.1007/BF00310320

Grell K. G., Ruthmann A. Placozoa. In: Microscopic Anatomy of Invertebrates. Vol. 2. Placozoa, Porifera, Cnidaria, and Ctenophora / F. W. Harrison, J. A. Westfall (Eds). New York : Wiley-Liss, 1991, pp. 13–28.

Gruber-Vodicka H. R., Leisch N., Kleiner M., Hinzke T., Liebeke M., McFall-Ngai M., Hadfield M. G., Dubilier N. Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2. Nature Microbiology, 2019, vol. 4, iss. 9, pp. 1465–1474. https://doi.org/10.1038/s41564-019-0475-9

Hardy S., Legagneux V., Audic Y., Paillard L. Reverse genetics in eukaryotes. Biology of the Cell, 2010, vol. 102, iss. 10, pp. 561–580. https://doi.org/10.1042/BC20100038

Harris A. K. Cell motility and the problem of anatomical homeostasis. In: Cell Behaviour: Shape, Adhesion and Motility. The Second Abercrombie Conf. [Proceed.] / S. E. Heaysman, C. A. Middleton, F. M. Watt (Eds). Cambridge : The Company of Biologists L., 1987, pp. 121–140. (Journal of Cell Science Supplements ; Suppl. 8). https://doi.org/10.1242/jcs.1987.Supplement_8.7

Heyland A., Croll R., Goodall S., Kranyak J., Russell W. Trichoplax adhaerens, an enigmatic basal metazoan with potential. In: Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocols / D. J. Carroll, S. A. Stricker (Eds). Totowa, NJ : Humana, 2014, pp. 45–61. https://doi.org/10.1007/978-1-62703-974-1_4

Jackson A. M., Buss L. W. Shiny spheres of placozoans (Trichoplax) function in anti-predator defense. Invertebrate Biology, 2009, vol. 128, iss. 3, pp. 205–212. https://doi.org/10.1111/J.1744-7410.2009.00177.X

Jakob W., Sagasser S., Dellaporta S., Holland P., Kuhn K., Schierwater B. The Trox-2 Hox/ParaHox gene of Trichoplax (Placozoa) marks an epithelial boundary. Development Genes and Evolution, 2004, vol. 214, iss. 4, pp. 170–175. https://doi.org/10.1007/s00427-004-0390-8

Kamm K., Osigus H. J., Stadler P. F., DeSalle R., Schierwater B. Trichoplax genomes reveal profound admixture and suggest stable wild populations without bisexual reproduction. Scientific Reports, 2018, vol. 8, iss. 1, art. no. 11168 (11 p.). https://doi.org/10.1038/s41598-018-29400-y

Kamm K., Schierwater B., DeSalle R. Innate immunity in the simplest animals – placozoans. BMC Genomics, 2019, vol. 20, iss. 1, art. no. 5 (12 p.). https://doi.org/10.1186/s12864-018-5377-3

Kuhl W., Kuhl G. Bewegungsphysiologische Untersuchungen an Trichoplax adhaerens F. E. Schulze. Zoologischer Anzeiger Supplement, 1963, vol. 26, pp. 460–469.

Kuhl W., Kuhl G. Untersuchungen über das Bewegungsverhalten von Trichoplax adhaerens F. E. Schulze (Zeittransformation: Zeitraffung). Zeitschrift für Morphologie und Ökologie der Tiere, 1966, vol. 56, iss. 4, pp. 417–435. https://doi.org/10.1007/BF00442291

Kuznetsov A. V., Halaimova A. V., Ufimtseva M. A., Chelebieva E. S. Blocking a chemical communication between Trichoplax organisms leads to their disorderly movement. International Journal of Parallel, Emergent and Distributed Systems, 2020b, vol. 35, iss. 4, pp. 473–482. https://doi.org/10.1080/17445760.2020.1753188

Layden M. J., Rentzsch F., Röttinger E. The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration. WIREs Developmental Biology, 2016, vol. 5, iss. 4, pp. 408–428. https://doi.org/10.1002/wdev.222

Lenhoff S. G., Lenhoff H. M. Hydra and the Birth of Experimental Biology, 1744: Abraham Trembley’s Memoires Concerning the Polyps. Pacific Grove, CA : Boxwood Press, 1986. 192 p.

Liu L.-P., Xiang J.-H., Dong B., Natarajan P., Yu K.-J., Cai N.-E. Ciona intestinalis as an emerging model organism: Its regeneration under controlled conditions and methodology for egg dechorionation. Journal of Zhejiang University SCIENCE B – Biomedicine & Biotechnology, 2006, vol. 7, iss. 6, pp. 467–474. https://doi.org/10.1631/jzus.2006.B0467

Lush M. E., Diaz D. C., Koenecke N., Baek S., Boldt H., St Peter M. K., Gaitan-Escudero T., Romero-Carvajal A., Busch-Nentwich E. M., Perera A. G., Hall K. E., Peak A., Haug J. S., Piotrowski T. scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling. eLife, 2019, vol. 25, art. no. e44431 (31 p.). https://doi.org/10.7554/eLife.44431

Mayorova T. D., Hammar K., Winters C. A., Reese T. S., Smith C. L. The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides. Biology Open, 2019, vol. 8, iss. 8, art. no. bio045674 (13 p.). https://doi.org/10.1242/bio.045674

Mayorova T. D., Smith C. L., Hammar K., Winters C. A., Pivovarova N. B., Aronova M. A., Leapman R. D., Reese T. S. Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses. PLoS One, 2018, vol. 13, iss. 1, art. no. e0190905 (20 p.). https://doi.org/10.1371/journal.pone.0190905

Moroz L. L., Sohn D., Romanova D. Y., Kohn A. B. Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate. Biochemical and Biophysical Research Communications, 2020, vol. 527, iss. 4, pp. 947–952. https://doi.org/10.1016/j.bbrc.2020.04.135

Pearse V. B. Growth and behavior of Trichoplax adhaerens: First record of the phylum Placozoa in Hawaii. Pacific Science, 1989, vol. 43, no. 2, pp. 117–121.

Pearse V. B., Voigt O. Field biology of placozoans (Trichoplax): Distribution, diversity, biotic interactions. Integrative & Comparative Biology, 2007, vol. 47, iss. 5, pp. 677–692. https://doi.org/10.1093/icb/icm015

Romanova D. Y., Heyland A., Sohn D., Kohn A. B., Fasshauer D., Varoqueaux F., Moroz L. L. Glycine as a signaling molecule and chemoattractant in Trichoplax (Placozoa): Insights into the early evolution of neurotransmitters. NeuroReport, 2020, vol. 31, iss. 6, pp. 490–497. https://doi.org/10.1097/WNR.0000000000001436

Ruthmann A. Cell differentiation, DNA content and chromosomes of Trichoplax adhaerens F. E. Schulze. Cytobiologie, 1977, vol. 15, iss. 1, pp. 58–64.

Ruthmann A., Terwelp U. Disaggregation and reaggregation of cells of the primitive metazoan Trichoplax adhaerens. Differentiation, 1979, vol. 13, iss. 3, pp. 185–198. https://doi.org/10.1111/j.1432-0436.1979.tb01581.x

Sambrook J., Russell D. Molecular Cloning: A Laboratory Manual. 3rd ed. New York : Cold Spring Harbor Laboratory Press, 2001, 2344 p.

Schierwater B., Eitel M., Jakob W., Osigus H. J., Hadrys H., Dellaporta S. L., Kolokotronis S. O., Desalle R. Concatenated analysis sheds light on early metazoan evolution and fuels a modern “urmetazoon” hypothesis. PLoS Biology, 2009, vol. 7, iss. 1, art. no. e1000020 (9 p.). https://doi.org/10.1371/journal.pbio.1000020

Schulze F. E. Trichoplax adhaerens, nov. gen., nov. spec. Zoologischer Anzeiger, 1883, vol. 6, no. 132, pp. 92–97.

Schulze F. E. Über Trichoplax adhaerens. Physikalische Abhandlungen der Königlichen Akademie der Wissenschaften zu Berlin, 1891, abh. 1, s. 1–23.

Schwartz V. Das radialpolare Differenzierungsmuster bei Trichoplax adhaerens F. E. Schulze (Placozoa). Zeitschrift für Naturforschung C, 1984, vol. 39, iss. 7–8, pp. 818–832. https://doi.org/10.1515/znc-1984-7-822

Sebé-Pedrós A., Chomsky E., Pang K., Lara-Astiaso D., Gaiti F., Mukamel Z., Amit I., Hejnol A., Degnan B. M., Tanay A. Early metazoan cell type diversity and the evolution of multicellular gene regulation. Nature Ecology & Evolution, 2018, vol. 2, iss. 7, pp. 1176–1188. https://doi.org/10.1038/s41559-018-0575-6

Senatore A., Reese T. S., Smith C. L. Neuropeptidergic integration of behavior in Trichoplax adhaerens, an animal without synapses. Journal of Experimental Biology, 2017, vol. 220, iss. 18, pp. 3381–3390. https://doi.org/10.1242/jeb.162396

Signorovitch A. Y., Buss L. W., Dellaporta S. L. Comparative genomics of large mitochondria in placozoans. PLoS Genetics, 2007, vol. 3, iss. 1, art. no. e13 (7 p.). https://doi.org/10.1371/journal.pgen.0030013

Smith C. L., Abdallah S., Wong Y. Y., Le P., Harracksingh A. N., Artinian L., Tamvacakis A. N., Rehder V., Reese T. S., Senatore A. Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue. Journal of General Physiology, 2017, vol. 149, no. 4, pp. 483–510. https://doi.org/10.1085/jgp.201611683

Smith C. L., Mayorova T. D. Insights into the evolution of digestive systems from studies of Trichoplax adhaerens. Cell and Tissue Research, 2019, vol. 377, iss. 3, pp. 353–367. https://doi.org/10.1007/s00441-019-03057-z

Smith C. L., Pivovarova N., Reese T. S. Coordinated feeding behavior in Trichoplax, an animal without synapses. PLoS One, 2015, vol. 10, iss. 9, art. no. e0136098 (15 p.). https://doi.org/10.1371/journal.pone.0136098

Smith C. L., Reese T. S., Govezensky T., Barrio R. A. Coherent directed movement toward food modeled in Trichoplax, a ciliated animal lacking a nervous system. Proceedings of the National Academy of Sciences, 2019, vol. 116, no. 18, pp. 8901–8908. https://doi.org/10.1073/pnas.1815655116

Smith C. L., Varoqueaux F., Kittelmann M., Azzam R. N., Cooper B., Winters C. A., Eitel M., Fasshauer D., Reese T. S. Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens. Current Biology, 2014, vol. 24, iss. 14, pp. 1565–1572. https://doi.org/10.1016/j.cub.2014.05.046

Sommer R. J. The future of evo-devo: Model systems and evolutionary theory. Nature Reviews Genetics, 2009, vol. 10, iss. 6, pp. 416–422. https://doi.org/10.1038/nrg2567

Srivastava M., Begovic E., Chapman J., Putnam N. H., Hellsten U., Kawashima T., Kuo A., Mitros T., Salamov A., Carpenter M. L., Signorovitch A. Y., Moreno M. A., Kamm K., Grimwood J., Schmutz J., Shapiro H., Grigoriev I. V., Buss L. W., Schierwater B., Dellaporta S. L., Rokhsar D. S. The Trichoplax genome and the nature of placozoans. Nature, 2008, vol. 454, iss. 7207, pp. 955–960. https://doi.org/10.1038/nature07191

Syed T., Schierwater B. Trichoplax adhaerens: Discovered as a missing link, forgotten as a hydrozoan, re-discovered as a key to metazoan evolution. Vie et Milieu, 2002, vol. 52, iss. 4, pp. 177–187.

Thiemann M., Ruthmann A. Alternative modes of asexual reproduction in Trichoplax adhaerens (Placozoa). Zoomorphology, 1991, vol. 110, iss. 3, pp. 165–174. https://doi.org/10.1007/BF01632872

Thiemann M., Ruthmann A. Trichoplax adhaerens F. E. Schulze (Placozoa): The formation of swarmers. Zeitschrift für Naturforschung C, 1988, vol. 43, iss. 11–12, pp. 955–957. https://doi.org/10.1515/znc-1988-11-1224

Transgenesis Techniques: Principles and Protocols. 3rd ed. / E. J. Cartwright (Ed.). Totowa, NJ : Humana Press, 2009, 335 p. https://doi.org/10.1007/978-1-60327-019-9

Varoqueaux F., Williams E. A., Grandemange S., Truscello L., Kamm K., Schierwater B., Jékely G., Fasshauer D. High cell diversity and complex peptidergic signaling underlie placozoan behavior. Current Biology, 2018, vol. 28, iss. 21, pp. 3495–3501. https://doi.org/10.1016/j.cub.2018.08.067

Wenderoth H. Transepithelial cytophagy by Trichoplax adhaerens F. E. Schulze (Placozoa) feeding on yeast. Zeitschrift für Naturforschung C, 1986, vol. 41, iss. 3, pp. 343–347. https://doi.org/10.1515/znc-1986-0316

Wilson H. V. Development of sponges from dissociated tissue cells. Fishery Bulletin, 1910, vol. 30, pp. 1–35.

Wilson H. V. On some phenomena of coalescence and regeneration in sponges. Journal of Experimental Zoology, 1907, vol. 5, iss. 2, pp. 245–258. https://doi.org/10.1002/jez.1400050204

Zuccolotto-Arellano J., Cuervo-González R. Binary fission in Trichoplax is orthogonal to the subsequent division plane. Mechanisms of Development, 2020, vol. 162, art. no. 103608 (9 p.). https://doi.org/10.1016/j.mod.2020.103608


File name: Supplementary-1
Description: Trichoplax 30 minutes after bisection
File extension: video/mp4
File name: Supplementary-2
Description: Trichoplax 60 minutes after bisection
File extension: video/mp4



Download data is not yet available.