The Crustacean Society © The Author(s) 2019. Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which per- mits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Culturing embryonic cells from the parthenogenetic clonal marble crayfish (Marmorkrebs) Procambarus virginalis Lyko, 2017 (Decapoda: Astacidea: Cambaridae) Heriberto DeLeon III 1 , Juan Garcia Jr. 1 , Dionn Carlo Silva 1 , Oscar Quintanilla 1 , Zen Faulkes 1 and John M. Thomas III 1,2,3 1 Department of Biology, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539, USA; 2 Center for Vector Borne Disease, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539, USA; and 3 School of Medicine, The University of Texas Rio Grande Valley, 1201 West University Drive, Edinburg, TX 78539, USA Correspondence: J. Thomas; e-mail: john.thomas@utrgv.edu (Received 13 March 2019; accepted 6 August 2019) ABSTRACT The parthenogenetic marbled crayfsh, or Marmorkrebs (Procambarus virginalis Lyko 2017), is an emerging model organism. We describe a method to isolate cells from early-stage embryos and culture them in vitro. The identity of the cells was confrmed by sequencing the cyto- chrome c oxidase subunit I (COI) gene. This technique can be applied for use in the manipu- lation of embryonic parthenogenetic crayfsh cells. Key Words: cell culture, embryos, invasive species, model organisms, ontogeny, techniques INTRODUCTION The marbled crayfsh or Marmorkrebs (Procambarus virginalis Lyko 2017 (Lyko, 2017a), formerly P. fallax f. virginalis (see Martin et al. 2010)), has many biologically signifcant features. It is the only obligate parthenogenetic decapod crustacean species (Scholtz et al., 2003) as a result of having a triploid genome (Vogt et al., 2015; Martin et al., 2016), which results in all individuals being genetically identical (Martin et al., 2007; Gutekunst et al., 2018). It has no known native habitat but it is a potential or actual in- vasive species that has been introduced on three continents and established populations in multiple countries (Hossain et al., 2018). Marmorkrebs were rated fourteenth on a list of top invasive spe- cies in Europe (Nentwig et al., 2018). The Marmorkrebs is also an emerging model organism in la- boratories (Vogt, 2008b; Jirikowski et al., 2010; Vogt, 2011; Faulkes, 2016; Hossain et al., 2018; Vogt, 2018a) in areas such as ethology (Hossain et al., 2019; Takahashi et al., 2019), develop- mental biology (Rieger & Harzsch, 2008; Shinji et al., 2019), epi- genetics (Gatzmann et al., 2018; Vogt, 2018b), toxicology (Naboka et al., 2018; Velisek et al., 2019), and cancer research (Vogt, 2008a; Lyko, 2017b). Marmorkrebs are the frst, and currently only, decapod crustacean species with a known complete genome se- quence (Gutekunst et al., 2018). Having a sequenced genome cre- ates possibilities to apply many genetic and molecular techniques that have long been applied in other model organisms, but only rarely with crayfshes. There are many pragmatic reasons to de- velop Marmorkrebs as a genetic model for crustaceans, notably understanding and managing its introduction and global spread as a non-native crayfsh species. One obstacle to using Marmorkrebs as a model for develop- mental biology and molecular biology is that the embryos con- tain large amounts of yolk, making eggs mostly opaque (Alwes & Scholtz, 2006). Imaging embryonic Marmorkrebs cells is dif- fcult compared to model organisms with transparent embryos, such as the amphipod Parhyale hawaiensis (Dana, 1853) (Browne et al., 2005), zebrafsh (Danio rerio (Hamilton, 1822)) (Lawrence, 2007), or tunicates (Miyamoto, 1985). Manipulation of em- bryonic cells in other models has also led to the development of transgenic animals (Hogan et al., 1994; Ofeld et al., 2000), which has furthered the utility of several animal models into multiple avenues of research. One possible solution would be to use cell culturing techniques (Toullec, 1999) to separate the developing cells from the yolk. Cells from adult crayfshes have been cultured successfully (Birmelin et al., 1998; Liu et al., 2011; Ding et al., 2012), but not from mature eggs. Embryonic cells have been cultured from shrimps (Penaeus spp.) (Toullec et al., 1996; Fan & Wang, 2002; Hu et al., 2008) and prawns (Macrobrachium rosenbergii De Man, 1879) (Frerichs, 1996), sug- gesting that culturing embryonic crayfsh cells is feasible. We re- port a technique for isolating and culturing cells derived from the mature eggs of P. virginalis. Journal of Crustacean Biology Advance Access published 26 September 2019 Journal of Crustacean Biology Journal of Crustacean Biology 39(6), 758–763, 2019. doi:10.1093/jcbiol/ruz063 Downloaded from https://academic.oup.com/jcb/article-abstract/39/6/758/5574472 by National Science and Technology Library -Root, john.thomas@utrgv.edu on 26 November 2019