Abstract
The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amparyup P, Charoensapsri W, Tassanakajon A (2013) Prophenoloxidase system and its role in shrimp immune responses against major pathogens. Fish Shellfish Immunol 34:990–1001. https://doi.org/10.1016/j.fsi.2012.08.019
Arockiaraj J, Easwvaran S, Vanaraja P et al (2012) Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infection on caspase 3c expression and activity in freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol 32:161–169. https://doi.org/10.1016/j.fsi.2011.11.006
Bravo R, Fey S, Bellatin J et al (1982) Identification of nuclear polypeptide (‘cyclin’) whose relative proportion is sensitive to changes in the rate of cell proliferation and to transformation. Prog Clin Biol Res 85:235–248
Chen SN, Wang CS (1999) Establishment of cell culture systems from penaeid shrimp and their susceptibility to white spot disease and yellow head viruses. Methods Cell Sci 21:199–206. https://doi.org/10.1023/A:1009885929335
Chen YY, Chen JC, Lin YC et al (2014) Shrimp that have received carrageenan via immersion and diet exhibit immunocompetence in phagocytosis despite a post-plateau in immune parameters. Fish Shellfish Immunol 36:352–366. https://doi.org/10.1016/j.fsi.2013.12.004
Coles J, Pipe R (1994) Phenoloxidase activity in the haemolymph and haemocytes of the marine mussel Mytilus edulis. Fish Shellfish Immunol 4:337–352
de-la-Re-Vega E, Muhlia-Almazan A, Arvizu-Flores AA et al (2011) Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection. Results Immunol 1:24–30. https://doi.org/10.1016/j.rinim.2011.07.001
Ellender R, Najafabadi A, Middlebrooks B (1992) Observations on the primary culture of hemocytes of Penaeus. J Crust Biol 12:178–185. https://doi.org/10.2307/1549072
Fan TJ, Wang XF (2002) In vitro culture of embryonic cells from the shrimp, Penaeus chinensis. J Exp Mar Biol Ecol 267:175–184. https://doi.org/10.1016/S0022-0981(01)00364-1 00364–1
Farook MA, Sundar Raj N, Madan N et al (2014) Immunomodulatory effect of recombinant Macrobrachium rosenbergii nodavirus capsid protein (r-MCP) against white tail disease of giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879). Aquaculture 433:395–403. https://doi.org/10.1016/j.aquaculture.2014.07.004
Frerichs GN (1996) In vitro culture of embryonic cells from the freshwater prawn Macrobrachium rosenbergii. Aquaculture 143:227–232. https://doi.org/10.1016/0044-8486(96)01281-1
Freshney RI (2005) Culture of animal cells: a manual of basic technique. Wiley, Hoboken
Ghnenis AB, Czaikowski RE, Zhang ZJ, Bushman JS (2018) Toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology. J Vis Exp 2018:1–7. https://doi.org/10.3791/58031
Goodman CL, El Sayed GN, Mcintosh AH et al (2001) Establishment and characterization of insect cell lines from 10 lepidopteran species. In Vitro Cell Dev Biol Animal 37:367–373. https://doi.org/10.1290/1071-2690(2001)037%3C0367:EACOIC%3E2.0.CO;2
Goswami M, Lakra WS, Rajaswaminathan T, Rathore G (2010) Development of cell culture system from the giant freshwater prawn Macrobrachium rosenbergii (de Man). Mol Biol Rep 37:2043–2048. https://doi.org/10.1007/s11033-009-9659-3
Harrison RL, Lynn DE (2008) New cell lines derived from the black cutworm, Agrotis ipsilon, that support replication of the A. ipsilon multiple nucleopolyhedrovirus and several group I nucleopolyhedroviruses. J Invertebr Pathol 99:28–34. https://doi.org/10.1016/j.jip.2008.02.015
Hu G-B, Wang D, Chen J-X (2010) Retroviral delivery of simian virus 40 large T antigen gene into primary cultured ovary cells of the penaeid shrimp, Penaeus chinensis: indirect evidence of retroviral integration. J World Aquac Soc 41:137–143. https://doi.org/10.1111/j.1749-7345.2009.00321.x
Hu G Bin, Wang D, Wang CH, Yang KF (2008) A novel immortalization vector for the establishment of penaeid shrimp cell lines. In Vitro Cell Dev Biol Animal 44:51–56. https://doi.org/10.1007/s11626-007-9076-7
Huang CC, Sritunyalucksana K, Söderhäll K, Song YL (2004) Molecular cloning and characterization of tiger shrimp (Penaeus monodon) transglutaminase. Dev Comp Immunol 28:279–294. https://doi.org/10.1016/j.dci.2003.08.005
Huang J, Li F, Wu J, Yang F (2015) White spot syndrome virus enters crayfish hematopoietic tissue cells via clathrin-mediated endocytosis. Virology 486:35–43. https://doi.org/10.1016/j.virol.2015.08.034
Jariyapong P, Pudgerd A, Cheloh N et al (2019) Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection. Fish Shellfish Immunol 86:756–763. https://doi.org/10.1016/j.fsi.2018.12.021
Jayesh P, Seena J, Singh ISB (2012) Establishment of shrimp cell lines: perception and orientation. Indian J Virol 23:244–251. https://doi.org/10.1007/s13337-012-0089-9
Jia Z, Kavungal S, Jiang S et al (2016) The characterization of hematopoietic tissue in adult Chinese mitten crab Eriocheir sinensis. Dev Comp Immunol 60:12–22. https://doi.org/10.1016/j.dci.2016.02.002
Jiravanichpaisal P, Söderhäll K, Söderhäll I (2004) Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish. Fish Shellfish Immunol 17:265–275. https://doi.org/10.1016/j.fsi.2004.03.010
Jiravanichpaisal P, Söderhäll K, Söderhäll I (2006) Characterization of white spot syndrome virus replication in in vitro-cultured haematopoietic stem cells of freshwater crayfish, Pacifastacus leniusculus. J Gen Virol 87:847–854. https://doi.org/10.1099/vir.0.81758-0
Johansson MW, Söderhäll K (1985) Exocytosis of the prophenoloxidase activating system from crayfish haemocytes. J Comp Physiol B 156:175–181. https://doi.org/10.1007/BF00695771
Kasornchandra J, Khongpradit R, Ekpanithanpong U, Boonyaratpalin S (1999) Progress in the development of shrimp cell cultures in Thailand. Methods Cell Sci 21:231–235. https://doi.org/10.1023/A:1009828632486
Leoni M, Manyuhina OV, Bowick MJ, Marchetti MC (2017) Defect driven shapes in nematic droplets: analogies with cell division. Soft Matter 13:1257–1266. https://doi.org/10.1039/C6SM02584F
Lin X, Söderhäll I (2011) Crustacean hematopoiesis and the astakine cytokines. Blood 117:6417–6424. https://doi.org/10.1182/blood-2010-11-320614
Lin X, Söderhäll K, Söderhäll I (2008) Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis. BMC Immunol 9:1–11. https://doi.org/10.1186/1471-2172-9-58
Lin X, Soderhall K, Soderhall I (2011) Invertebrate hematopoiesis: an astakine-dependent novel hematopoietic factor. J Immunol 186:2073–2079. https://doi.org/10.4049/jimmunol.1001229
Ling E, Shirai K, Kanehatsu R, Kiguchi K (2005) Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori. Tissue Cell 37:101–107. https://doi.org/10.1016/J.TICE.2004.10.007
Liu CH, Tseng DY, Lai CY et al (2006a) Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage. Fish Shellfish Immunol 21:60–69. https://doi.org/10.1016/j.fsi.2005.10.004
Liu G, Yang L, Fan T et al (2006b) Purification and characterization of phenoloxidase from crab Charybdis japonica. Fish Shellfish Immunol 20:47–57. https://doi.org/10.1016/j.fsi.2005.03.012
Ma J, Zeng L, Lu Y (2017) Penaeid shrimp cell culture and its applications. Rev Aquac 9:88–98. https://doi.org/10.1111/raq.12106
Muhammad F, Zhang ZF, Shao MY et al (2013) Genesis of hematopoietic tissue and its relation with hemocytes of litopenaeus vannamei (boone, 1931) (crustacea: Decapoda). Pak Vet J 33:91–95
Nappi A, Seymour J (1991) Hemolymph phenol oxidases in Drosophila melanogaster, Locusta migratoria, and Austropotamobius pallipes. Biochem Biophys Res Commun 180:748–754
Owens L, Smith J (1999) Early attempts at production of prawn cell lines. Methods Cell Sci 21:207–211. https://doi.org/10.1023/A:1009806606562
Pillai D, Bonami JR (2012) A review on the diseases of freshwater prawns with special focus on white tail disease of Macrobrachium rosenbergii. Aquacult Res 43:1029–1037
Pudgerd A, Chotwiwatthanakun C, Kruangkum T et al (2019) The hematopoietic organ of Macrobrachium rosenbergii: structure, organization and immune status. Fish Shellfish Immunol 88:415–423. https://doi.org/10.1016/j.fsi.2019.03.011
Puthumana J, Jose S, Philip R, Singh ISB (2015) Cellular and molecular markers in monitoring the fate of lymphoid cell culture from Penaeus monodon Fabricius (1798). Fish Shellfish Immunol 47:893–901. https://doi.org/10.1016/j.fsi.2015.05.033
Puthumana J, Philip R, Bright Singh IS (2016) Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters. Cytotechnology 68:1147–1159. https://doi.org/10.1007/s10616-015-9872-y
Ramírez-Clavijo S, Montoya-Ortíz G (2013) Gene expression and regulation. In: Anaya J-M, Shoenfeld Y, Rojas-Villarraga A et al (eds) Autoimmunity: from bench to bedside, 1st edn. El Rosario University Press, Bogota, p 3
Ryazanova AD, Alekseev AA, Slepneva IA (2012) The phenylthiourea is a competitive inhibitor of the enzymatic oxidation of DOPA by phenoloxidase. J Enzyme Inhib Med Chem 27:78–83. https://doi.org/10.3109/14756366.2011.576010
Shi H, Ruan L, Söderhäll I et al (2018) Transfection of crayfish hematopoietic tissue cells. Dev Comp Immunol 88:70–76. https://doi.org/10.1016/j.dci.2018.07.005
Smith VJ, Söderhäll K (1983) Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro. Cell Tissue Res 233:295–303. https://doi.org/10.1007/BF00238297
Söderhäll I (2013) Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity. Cytotechnology 65:691–695. https://doi.org/10.1007/s10616-013-9578-y
Söderhäll I, Bangyeekhun E, Mayo S, Söderhäll K (2003) Hemocyte production and maturation in an invertebrate animal; proliferation and gene expression in hematopoietic stem cells of Pacifastacus leniusculus. Dev Comp Immunol 27:661–672. https://doi.org/10.1016/S0145-305X(03)00039-9
Soderhall I, Kim Y-A, Jiravanichpaisal P et al (2005) An ancient role for a prokineticin domain in invertebrate hematopoiesis. J Immunol 174:6153–6160. https://doi.org/10.4049/jimmunol.174.10.6153
Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28. https://doi.org/10.1016/S0952-7915(98)80026-5
Söderhäll K, Smith V (1983) Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Dev Comp Immunol 7:229–239
Telentschak S, Soliwoda M, Nohroudi K et al (2015) Cytokinesis failure and successful multipolar mitoses drive aneuploidy in glioblastoma cells. Oncol Rep 33:2001–2008. https://doi.org/10.3892/or.2015.3751
Thansa K, Rungsiwiwut R, Kitiyanant N, Taengchaiyaphum S (2018) Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon). Fish Shellfish Immunol 81:204–213. https://doi.org/10.1016/j.fsi.2018.07.030
Thansa K, Yocawibun P, Suksodsai H (2016) The cellular death pattern of primary haemocytes isolated from the black tiger shrimp (Penaeus monodon). Fish Shellfish Immunol. https://doi.org/10.1016/j.fsi.2016.08.045
Toullec JY (1999) Crustacean primary cell culture: a technical approach. Methods Cell Sci 21:193–198. https://doi.org/10.1023/A:1009833924792
Winstanley D, Crook NE (1993) Replication of Cydia pomonella granulosis virus in cell cultures. J Gen Virol 74:1599–1609. https://doi.org/10.1099/0022-1317-74-8-1599
Wu J, Li F, Huang J et al (2015) Crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication. Fish Shellfish Immunol 43:67–74. https://doi.org/10.1016/j.fsi.2014.12.013
Xie Y, Wang B, Li F et al (2008) Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis. Comp Biochem Physiol B 151:225–229. https://doi.org/10.1016/j.cbpb.2008.07.006
Xing J, Bin ZW, Zhou L (2002) Endoenzymes associated with haemocyte types in the scallop (Chlamys farreri). Fish Shellfish Immunol 13:271–278. https://doi.org/10.1006/fsim.2001.0402
Xu X, Duan H, Shi Y et al (2018) Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods. Dev Comp Immunol 88:45–54. https://doi.org/10.1016/j.dci.2018.07.006
Acknowledgements
We would like to express our thanks to Mahidol University for supporting our work. Our sincere thanks also go to the staff and students at the Faculty of Sciences, Mahidol University and the Integrative Aquaculture Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand, for their support. Finally, our special thanks to Prof. Timothy Flegel for his comments and proofreading of this article.
Author information
Authors and Affiliations
Contributions
Conceptualization: KT, TK, CC; methodology: KT, TK, AP, PA; validation: KT, AP, LC, PA, RS; formal analysis: KT, AP, LC, PA, RS; investigation: KT, AP, LC, PA, RS; resources: KT, TK, PA, CC; writing—original draft: KT; writing—review and editing: KT, TK, AP, PA, RS, CC, RV, KS; supervision: KT, TK, CC, RV, KS; project administration: KT, TK; funding acquisition: KT, TK, PA, CC, RV, KS.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest to disclose.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Thansa, K., Kruangkum, T., Pudgerd, A. et al. Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii. Cytotechnology 73, 141–157 (2021). https://doi.org/10.1007/s10616-021-00451-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-021-00451-w