Eiler and colleagues 2025

Eiler A, Stensrud E, Osman O. 2025. Detection of marbled crayfish Procambarus fallax. protocols.io. https://dx.doi.org/10.17504/protocols.io.q26g78199lwz/v1

 

Abstract

Taqman QPCR assay for marbled crayfish Procambarus fallax.

 

Keywords:  None provided.

 

DeLeon and colleagues 2019

DeLeon H III, Garcia J Jr., Silva DC, Quintanilla O, Faulkes Z, Thomas JM III. 2019. Culturing embryonic cells from the parthenogenetic clonal marble crayfish (Marmorkrebs) Procambarus virginalis Lyko, 2017 (Decapoda: Astacidea: Cambaridae). Journal of Crustacean Biology 39(6): 758–763. https://doi.org/10.1093/jcbiol/ruz063.

Abstract

The parthenogenetic marbled crayfish, 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 confirmed by sequencing the cytochrome c oxidase subunit I (COI) gene. This technique can be applied for use in the manipulation of embryonic parthenogenetic crayfish cells.

Keywords: cell culture • embryos • invasive species • model organisms • ontogeny • techniques

Gutekunst and colleagues, 2015

Gutekunst J, Falckenhayn C, Raddatz G, Lyko F. 2015. Assembly and annotation of the marbled crayfish genome. Poster presented Ninth Annual Arthropod Genomics Symposium, Kansas State University, Manhattan, Kansas, 17-19 June 2015. Abstract and program book, p. 23. http://www.k-state.edu/agc/images/symposium/Abstracts-ProgramBook.pdf

Abstract

Marbled crayfish are the only freshwater crayfish known to reproduce by cloning (apomictic parthenogenesis). Notably, among genetically identical offspring raised in the same environment phenotypic differences can be observed. Such non-genomic characteristics render the marbled crayfish an interesting laboratory model, especially for the field of epigenetics. We experimentally determined the genome size at approximately 3.8 Gbp by k-mer analysis and flow cytometry. Two individual females (Koelle, Steuerwald) were sequenced using shotgun sequencing with various insert sizes generating 350 Gbp and 196 Gbp of data respectively. High coverage sequencing data of Koelle was used to produce a first de novo draft assembly with a length weighted median contig size (N50) of 809 bp and scaffold N50 of 41 kb. To unambiguously demonstrate clonal reproduction in the marbled crayfish we are currently evaluating sequencing data from the second individual (Steuerwald). Transcriptome data provides additional information for quality control and assembly refinement. Genome wide comparisons to other arthropods will allow us to define characteristic features of decapods as ecologically and economically keystone species.

Keywords: None provided

Transgenic methods

One of the exciting prospects of Marmorkrebs is that any genetic change to an individual should be propagated to all its offspring, so changes introduced by mutations and transgenics would be easily maintained.

Vogt and colleagues (2004) note that Sarmasik and collegues (2001) had successfully transformed the crayfish species Procambarus clarkii. Sarmasik and colleagues based their methods on those of Burns and colleagues (1993) and Yee and colleagues (1994). All of which tells you how to design this stuff from scratch.

Fortunately, this does not appear to be necessary. A pantropic retroviral expression system is available commercially from ClonTech, which seems to be based on the work of Burns and colleagues.

Note, however, that because this is a viral method of delivery, it has a higher biosafety level than many labs might have.

References

Burns JC, Friedmann T, Driever W, Burrascano M, Yee J. 1993. Vesicular stomatitis Virus G glycoprotein pseudotyped retroviral vectors: Concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proceedings of the National Academy of Sciences 90(17): 8033-8037.
http://www.pnas.org/cgi/content/abstract/90/17/8033

Sarmasik A, Chun CZ, Jang I-K, Lu JK, Chen TT. 2001. Production of transgenic live-bearing fish and crustaceans with replication-defective pantropic retroviral vectors. Marine Biotechnology 3: S177-S184.
http://dx.doi.org/10.1007/s10126-001-0040-3

Sarmasik A, Jang I-K, Chun CZ, Lu JK, Chen TT. 2001. Transgenic live-bearing fish and crustaceans produced by transforming immature gonads with replication-defective pantropic retroviral vectors. Marine Biotechnology 3(5): 470-477.
http://dx.doi.org/10.1007/s10126001-0019-0

Yee J, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T. 1994. A General method for the generation of high-titer, pantropic retroviral vectors: Highly efficient infection of primary hepatocytes. Proceedings of the National Academy of Sciences 91(20): 9564-9568.
http://dx.doi.org/10.1073/pnas.91.20.9564