Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Multigenerational laboratory culture of pelagic ctenophores and CRISPR–Cas9 genome editing in the lobate Mnemiopsis leidyi

Abstract

Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi. A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR–Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Body plan of Mnemiopsis leidyi with major axes labeled.
Fig. 2: Equipment used for housing, feeding, spawning and general tank maintenance.
Fig. 3: Mnemiopsis leidyi embryogenesis.
Fig. 4: Stages of post-hatching development in Mnemiopsis leidyi.
Fig. 5: Equipment used for maintaining Brachionus plicatilis rotifer cultures.
Fig. 6: Microinjection.
Fig. 7: Pharynx formation is disrupted in Mnemiopsis leidyi by Bra sgRNA/Cas9 microinjection.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

References

  1. Dunn, C. W., Leys, S. P. & Haddock, S. H. D. The hidden biology of sponges and ctenophores. Trends Ecol. Evol. 30, 282–291 (2015).

    PubMed  Article  Google Scholar 

  2. Neff, E. P. What is a lab animal? Lab Anim. 47, 223–227 (2018).

    Article  Google Scholar 

  3. Ryan, J. F., Schnitzler, C. E. & Tamm, S. L. Meeting report of Ctenopalooza: the first international meeting of ctenophorologists. Evodevo 7, 19 (2016).

    PubMed Central  Article  Google Scholar 

  4. Chun, C. Die Ctenophoren des Golfes von Neapel und der angrenzenden Meeres-Abschnitte. (W. Engelmann, 1880).

  5. Hyman, L. H. in The Invertebrates: Protozoa through Ctenophora vol. 1 662–695 (McGraw-Hill, 1940).

  6. Harbison, G. R., Madin, L. P. & Swanberg, N. R. On the natural history and distribution of oceanic ctenophores. Deep Sea Res. I 25, 233–256 (1978).

    Article  Google Scholar 

  7. Harbison, G. R. in The Origins and Relationships of Lower Invertebrates (eds. Morris, S. C., George, J. D., Gibson, R. & Platt, H. M.) 78–100 (Oxford Univ. Press, 1985).

  8. Mills, C. E. & Haddock, S. H. D. in Light and Smith’s Manual: Intertidal Invertebrates of the Central California Coast (ed. Carlton, J. T.) 47–49 (Univ. California Press, 2007).

  9. Pang, K. & Martindale, M. Q. Ctenophores. Curr. Biol. 18, R1119–R1120 (2008).

    CAS  PubMed  Article  Google Scholar 

  10. Dunn, C. W. et al. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452, 745–749 (2008).

    CAS  PubMed  Article  Google Scholar 

  11. Hejnol, A. et al. Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc. Biol. Sci. 276, 4261–4270 (2009).

    PubMed  PubMed Central  Google Scholar 

  12. Philippe, H. et al. Phylogenomics revives traditional views on deep animal relationships. Curr. Biol. 19, 706–712 (2009).

    CAS  PubMed  Article  Google Scholar 

  13. Pick, K. S. et al. Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships. Mol. Biol. Evol. 27, 1983–1987 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Ryan, J. F. et al. The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342, 1242592 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  15. Moroz, L. L. et al. The ctenophore genome and the evolutionary origins of neural systems. Nature 510, 109–114 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Pisani, D. et al. Genomic data do not support comb jellies as the sister group to all other animals. Proc. Natl. Acad. Sci. USA. 112, 15402–15407 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Telford, M. J., Budd, G. E. & Philippe, H. Phylogenomic insights into animal evolution. Curr. Biol. 25, R876–R887 (2015).

    CAS  PubMed  Article  Google Scholar 

  18. Shen, X.-X., Hittinger, C. T. & Rokas, A. Contentious relationships in phylogenomic studies can be driven by a handful of genes. Nat. Ecol. Evol. 1, 126 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  19. Whelan, N. V. et al. Ctenophore relationships and their placement as the sister group to all other animals. Nat. Ecol. Evol. 1, 1737–1746 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  20. Li, Y., Shen, X.-X., Evans, B., Dunn, C. W. & Rokas, A. Rooting the animal tree of life. Mol. Biol. Evol. 38, 4322–4333 (2021).

    PubMed  PubMed Central  Article  Google Scholar 

  21. Afzelius, B. A. The fine structure of the cilia from ctenophore swimming-plates. J. Biophys. Biochem. Cytol. 9, 383–394 (1961).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Tamm, S. L. Mechanisms of ciliary co-ordination in ctenophores. J. Exp. Biol. 59, 231–245 (1973).

    Article  Google Scholar 

  23. Tamm, S. L. Cilia and the life of ctenophores. Invertebr. Biol. 133, 1–46 (2014).

    Article  Google Scholar 

  24. Abbott, J. F. The morphology of Coeloplana. Zool. Jahrb. Abt. Anat. Ontog. Tiere 24, 41–70 (1907).

    Google Scholar 

  25. Bargmann, W., Jacob, K. & Rast, A. Über Tentakel und Colloblasten der Ctenophore Pleurobrachia pileus. Z. Zellforsch. 123, 121–152 (1972).

    CAS  PubMed  Article  Google Scholar 

  26. von Byern, J., Mills, C. E. & Flammang, P. in Biological Adhesive Systems: From Nature to Technical and Medical Application (eds. von Byern, J. & Grunwald, I.) 29–40 (Springer, 2010).

  27. Leonardi, N. D., Thuesen, E. V. & Haddock, S. H. D. A sticky thicket of glue cells: a comparative morphometric analysis of colloblasts in 20 species of comb jelly (phylum Ctenophora). Cienc. Mar. 46, 211–225 (2020).

    CAS  Article  Google Scholar 

  28. Horridge, G. A. Relations between nerves and cilia in ctenophores. Am. Zool. 5, 357–375 (1965).

    CAS  PubMed  Article  Google Scholar 

  29. Tamm, S. L. Formation of the statolith in the ctenophore Mnemiopsis leidyi. Biol. Bull. 227, 7–18 (2014).

    PubMed  Article  Google Scholar 

  30. Jokura, K. & Inaba, K. Structural diversity and distribution of cilia in the apical sense organ of the ctenophore Bolinopsis mikado. Cytoskeleton 77, 442–455 (2020).

    CAS  PubMed  Article  Google Scholar 

  31. Jager, M. et al. New insights on ctenophore neural anatomy: immunofluorescence study in Pleurobrachia pileus (Müller, 1776). J. Exp. Zool. B 316B, 171–187 (2011).

    Article  Google Scholar 

  32. Moroz, L. L. & Kohn, A. B. Independent origins of neurons and synapses: insights from ctenophores. Philos. Trans. R. Soc. Lond. B 371, 20150041 (2016).

    Article  CAS  Google Scholar 

  33. Horridge, G. A. The giant mitochondria of ctenophore comb-plates. J. Cell Sci. s3-105, 301–310 (1964).

    Article  Google Scholar 

  34. Pett, W. et al. Extreme mitochondrial evolution in the ctenophore Mnemiopsis leidyi: insight from mtDNA and the nuclear genome. Mitochondrial DNA 22, 130–142 (2011).

    CAS  PubMed  Article  Google Scholar 

  35. Kohn, A. B. et al. Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei. Mol. Phylogenet. Evol. 63, 203–207 (2012).

    CAS  PubMed  Article  Google Scholar 

  36. Christianson, L. M., Johnson, S. B., Schultz, D. T. & Haddock, S. H. D. Hidden diversity of Ctenophora revealed by new mitochondrial COI primers and sequences. Mol. Ecol. Resour. 22, 283–294 (2022).

    CAS  PubMed  Article  Google Scholar 

  37. Hernandez-Nicaise, M. L., Mackie, G. O. & Meech, R. W. Giant smooth muscle cells of Beroë. Ultrastructure, innervation, and electrical properties. J. Gen. Physiol. 75, 79–105 (1980).

    CAS  PubMed  Article  Google Scholar 

  38. Hernandez-Nicaise, M. L. & Amsellem, J. Ultrastructure of the giant smooth muscle fiber of the ctenophore Beroe ovata. J. Ultrastruct. Res. 72, 151–168 (1980).

    CAS  PubMed  Article  Google Scholar 

  39. Hernandez-Nicaise, M.-L., Nicaise, G. & Malaval, L. Giant smooth muscle fibers of the ctenophore Mnemiopsis leidyi: ultrastructural study of in situ and isolated cells. Biol. Bull. 167, 210–228 (1984).

    Article  Google Scholar 

  40. Mackie, G. O., Mills, C. E. & Singla, C. L. Structure and function of the prehensile tentilla of Euplokamis (Ctenophora, Cydippida). Zoomorphology 107, 319–337 (1988).

    Article  Google Scholar 

  41. Vandepas, L. E., Warren, K. J., Amemiya, C. T. & Browne, W. E. Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi. J. Exp. Biol. 220, 1197–1201 (2017).

    PubMed  Google Scholar 

  42. Presnell, J. S. et al. The presence of a functionally tripartite through-gut in Ctenophora has implications for metazoan character trait evolution. Curr. Biol. 26, 2814–2820 (2016).

    CAS  PubMed  Article  Google Scholar 

  43. Haddock, S. H. D. & Case, J. F. Not all ctenophores are bioluminescent: Pleurobrachia. Biol. Bull. 189, 356–362 (1995).

    CAS  PubMed  Article  Google Scholar 

  44. Bessho-Uehara, M. et al. Evidence for de novo biosynthesis of the luminous substrate coelenterazine in ctenophores. iScience 23, 101859 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Martindale, M. Q. The onset of regenerative properties in ctenophores. Curr. Opin. Genet. Dev. 40, 113–119 (2016).

    CAS  PubMed  Article  Google Scholar 

  46. Edgar, A., Mitchell, D. G. & Martindale, M. Q. Whole-body regeneration in the lobate ctenophore Mnemiopsis leidyi. Genes 12, (2021).

  47. Ramon-Mateu, J., Ellison, S. T., Angelini, T. E. & Martindale, M. Q. Regeneration in the ctenophore Mnemiopsis leidyi occurs in the absence of a blastema, requires cell division, and is temporally separable from wound healing. BMC Biol. 17, 80 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  48. Chun, C. Die Dissogonie, eine neue Form der geschlechtlichen Zeugung. Festsch. zum siebensigsten Geburtstage Rudorf Leuckarts. Engelmarm, Leipzig 77–108 (1892).

  49. Martindale, M. Q. Larval reproduction in the ctenophore Mnemiopsis mccradyi (order Lobata). Mar. Biol. 94, 409–414 (1987).

    Article  Google Scholar 

  50. Hirota, J. Laboratory culture and metabolism of the planktonic ctenophore, Pleurobrachia bachei A. Agassiz. in Biological oceanography of the northern North Pacific Ocean (ed. Takenouti, A. Y.) 465–484 (Idemitu Shoten, 1972).

  51. Edgar, A., Ponciano, J. M. & Martindale, M. Q. Ctenophores are direct developers that reproduce continuously beginning very early after hatching. Proc. Natl Acad. Sci. 119, e2122052119 (2022).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Agassiz, A. Embryology of the Ctenophorae. Mem. Am. Acad. Arts Sci. 10, 357–398 (1874).

    Google Scholar 

  53. Hertwig, R. Über den Bau der Ctenophoren (Fischer, G, 1880).

  54. Driesch, H. & Morgan, T. H. Zur Analysis der ersten Entwickelungsstadien des Ctenophoreneies. Wilhelm. Roux Arch. Entwickl. Mech. Org. 2, 204–215 (1895).

    Google Scholar 

  55. Fischel, A. Experimentelle Untersuchungen am Ctenophorenei. Arch. Entwickelungsmech. Organismen 6, 109–130 (1897).

    Article  Google Scholar 

  56. Yatsu, N. Observations and experiments on the ctenophore egg: II. Notes on early cleavage stages and experiments on cleavage. Annot. Zool. Jpn 7, 333–346 (1911).

    Google Scholar 

  57. Podar, M., Haddock, S. H., Sogin, M. L. & Harbison, G. R. A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes. Mol. Phylogenet. Evol. 21, 218–230 (2001).

    CAS  PubMed  Article  Google Scholar 

  58. Simion, P., Bekkouche, N., Jager, M., Quéinnec, E. & Manuel, M. Exploring the potential of small RNA subunit and ITS sequences for resolving phylogenetic relationships within the phylum Ctenophora. Zoology 118, 102–114 (2015).

    PubMed  Article  Google Scholar 

  59. Yatsu, N. Observations and experiments on the ctenophore egg: III. Experiments on germinal localization of the egg of Beroe ovata. Annot. Zool. Jpn 8, 5–13 (1912).

    Google Scholar 

  60. Franc, J.-M. Etude ultrastructurale de la spermatogenèse du Cténaire Beroe ovata. J. Ultrastruct. Res. 42, 255–267 (1973).

    CAS  PubMed  Article  Google Scholar 

  61. Carré, D. & Sardet, C. Fertilization and early development in Beroe ovata. Dev. Biol. 105, 188–195 (1984).

    PubMed  Article  Google Scholar 

  62. Carré, D., Rouvière, C. & Sardet, C. In vitro fertilization in ctenophores: sperm entry, mitosis, and the establishment of bilateral symmetry in Beroe ovata. Dev. Biol. 147, 381–391 (1991).

    PubMed  Article  Google Scholar 

  63. Goudeau, M. & Goudeau, H. Successive electrical responses to insemination and concurrent sperm entries in the polyspermic egg of the ctenophore Beroe ovata. Dev. Biol. 156, 537–551 (1993).

    CAS  PubMed  Article  Google Scholar 

  64. Houliston, E., Carré, D., Johnston, J. A. & Sardet, C. Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata. Development 117, 75–87 (1993).

    CAS  PubMed  Article  Google Scholar 

  65. Rouvière, C., Houliston, E., Carré, D., Chang, P. & Sardet, C. Characteristics of pronuclear migration in Beroe ovata. Cell Motil. Cytoskelet. 29, 301–311 (1994).

    Article  Google Scholar 

  66. Jokura, K. et al. CTENO64 is required for coordinated paddling of ciliary comb plate in ctenophores. Curr. Biol. 29, 3510–3516.e4 (2019).

    CAS  PubMed  Article  Google Scholar 

  67. Derelle, R. & Manuel, M. Ancient connection between NKL genes and the mesoderm? Insights from Tlx expression in a ctenophore. Dev. Genes Evol. 217, 253–261 (2007).

    CAS  PubMed  Article  Google Scholar 

  68. Jager, M., Quéinnec, E., Chiori, R., Le Guyader, H. & Manuel, M. Insights into the early evolution of SOX genes from expression analyses in a ctenophore. J. Exp. Zool. B 310, 650–667 (2008).

    Article  CAS  Google Scholar 

  69. Alié, A. et al. Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of “germline genes” with stemness. Dev. Biol. 350, 183–197 (2011).

    PubMed  Article  CAS  Google Scholar 

  70. Dayraud, C. et al. Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: a ctenophore perspective. BMC Evol. Biol. 12, 107 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. Jager, M. et al. Evidence for involvement of Wnt signalling in body polarities, cell proliferation, and the neuro-sensory system in an adult ctenophore. PLoS ONE 8, e84363 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  72. Freeman, G. The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores. Dev. Biol. 51, 332–337 (1976).

    CAS  PubMed  Article  Google Scholar 

  73. Freeman, G. The role of cleavage in the localization of developmental potential in the ctenophore Mnemiopsis leidyi. Dev. Biol. 49, 143–177 (1976).

    CAS  PubMed  Article  Google Scholar 

  74. Freeman, G. The establishment of the oral–aboral axis in the ctenophore embryo. Development 42, 237–260 (1977).

    Article  Google Scholar 

  75. Martindale, M. Q. The ontogeny and maintenance of adult symmetry properties in the ctenophore, Mnemiopsis mccradyi. Dev. Biol. 118, 556–576 (1986).

    CAS  PubMed  Article  Google Scholar 

  76. Martindale, M. Q. & Henry, J. Q. Diagonal development: establishment of the anal axis in the ctenophore Mnemiopsis leidyi. Biol. Bull. 189, 190–192 (1995).

    CAS  PubMed  Article  Google Scholar 

  77. Martindale, M. Q. & Henry, J. Q. Development and regeneration of comb plates in the ctenophore Mnemiopsis leidyi. Biol. Bull. 191, 290–292 (1996).

    CAS  PubMed  Article  Google Scholar 

  78. Martindale, M. Q. & Henry, J. Q. Reassessing embryogenesis in the Ctenophora: the inductive role of e1 micromeres in organizing ctene row formation in the “mosaic” embryo, Mnemiopsis leidyi. Development 124, 1999–2006 (1997).

    CAS  PubMed  Article  Google Scholar 

  79. Martindale, M. Q. & Henry, J. Q. Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages. Dev. Biol. 214, 243–257 (1999).

    CAS  PubMed  Article  Google Scholar 

  80. Henry, J. Q. & Martindale, M. Q. Regulation and regeneration in the ctenophore Mnemiopsis leidyi. Dev. Biol. 227, 720–733 (2000).

    CAS  PubMed  Article  Google Scholar 

  81. Henry, J. Q. & Martindale, M. Q. Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi. Dev. Biol. 238, 40–46 (2001).

    CAS  PubMed  Article  Google Scholar 

  82. Henry, J. Q. & Martindale, M. Q. Inductive interactions and embryonic equivalence groups in a basal metazoan, the ctenophore Mnemiopsis leidyi. Evol. Dev. 6, 17–24 (2004).

    PubMed  Article  Google Scholar 

  83. Fischer, A. H., Pang, K., Henry, J. Q. & Martindale, M. Q. A cleavage clock regulates features of lineage-specific differentiation in the development of a basal branching metazoan, the ctenophore Mnemiopsis leidyi. Evodevo 5, 4 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  84. Babonis, L. S. et al. Integrating embryonic development and evolutionary history to characterize tentacle-specific cell types in a ctenophore. Mol. Biol. Evol. 35, 2940–2956 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Yamada, A. & Martindale, M. Q. Expression of the ctenophore Brain Factor 1 forkhead gene ortholog (ctenoBF-1) mRNA is restricted to the presumptive mouth and feeding apparatus: implications for axial organization in the Metazoa. Dev. Genes Evol. 212, 338–348 (2002).

    CAS  PubMed  Article  Google Scholar 

  86. Yamada, A., Pang, K., Martindale, M. Q. & Tochinai, S. Surprisingly complex T-box gene complement in diploblastic metazoans. Evol. Dev. 9, 220–230 (2007).

    CAS  PubMed  Article  Google Scholar 

  87. Pang, K. & Martindale, M. Q. Developmental expression of homeobox genes in the ctenophore Mnemiopsis leidyi. Dev. Genes Evol. 218, 307–319 (2008).

    CAS  PubMed  Article  Google Scholar 

  88. Layden, M. J., Meyer, N. P., Pang, K., Seaver, E. C. & Martindale, M. Q. Expression and phylogenetic analysis of the zic gene family in the evolution and development of metazoans. Evodevo 1, 12 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. Pang, K. et al. Genomic insights into Wnt signaling in an early diverging metazoan, the ctenophore Mnemiopsis leidyi. Evodevo 1, 10 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. Pang, K., Ryan, J. F., Baxevanis, A. D. & Martindale, M. Q. Evolution of the TGF-β signaling pathway and its potential role in the ctenophore, Mnemiopsis leidyi. PLoS ONE 6, e24152 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  91. Reitzel, A. M. et al. Nuclear receptors from the ctenophore Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: lineage-specific loss or ancestral condition in the emergence of the nuclear receptor superfamily? Evodevo 2, 3 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  92. Schnitzler, C. E. et al. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes. BMC Biol. 10, 107 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. Simmons, D. K., Pang, K. & Martindale, M. Q. Lim homeobox genes in the ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification. Evodevo 3, 2 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  94. Schnitzler, C. E., Simmons, D. K., Pang, K., Martindale, M. Q. & Baxevanis, A. D. Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation. Evodevo 5, 15 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  95. Reitzel, A. M., Pang, K. & Martindale, M. Q. Developmental expression of “germline”- and “sex determination”-related genes in the ctenophore Mnemiopsis leidyi. Evodevo 7, 17 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  96. Presnell, J. S. & Browne, W. E. Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing. Development 148, dev199771 (2021).

    CAS  PubMed  Article  Google Scholar 

  97. Lowe, S., Browne, M., Boudjelas, S. & De Poorter, M. 100 of the World’s Worst Invasive Alien Species: A Selection from the Global Invasive Species Database (Hollands Printing, 2000).

  98. Kideys, A. E. Ecology. Fall and rise of the Black Sea ecosystem. Science 297, 1482–1484 (2002).

    CAS  PubMed  Article  Google Scholar 

  99. Costello, J. H., Bayha, K. M., Mianzan, H. W., Shiganova, T. A. & Purcell, J. E. Transitions of Mnemiopsis leidyi (Ctenophora: Lobata) from a native to an exotic species: a review. Hydrobiologia 690, 21–46 (2012).

    Article  Google Scholar 

  100. Jaspers, C. et al. Ocean current connectivity propelling the secondary spread of a marine invasive comb jelly across western Eurasia. Glob. Ecol. Biogeogr. 27, 814–827 (2018).

    Article  Google Scholar 

  101. Jaspers, C. et al. Invasion genomics uncover contrasting scenarios of genetic diversity in a widespread marine invader. Proc. Natl Acad. Sci. USA 118, e2116211118 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. Colin, S. P., Costello, J. H., Hansson, L. J., Titelman, J. & Dabiri, J. O. Stealth predation and the predatory success of the invasive ctenophore Mnemiopsis leidyi. Proc. Natl Acad. Sci. USA 107, 17223–17227 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  103. Gemmell, B. J., Colin, S. P., Costello, J. H. & Sutherland, K. R. A ctenophore (comb jelly) employs vortex rebound dynamics and outperforms other gelatinous swimmers. R. Soc. Open Sci. 6, 181615 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  104. Jaspers, C., Titelman, J., Hansson, L. J., Haraldsson, M. & Ditlefsen, C. R. The invasive ctenophore Mnemiopsis leidyi poses no direct threat to Baltic cod eggs and larva. Limnol. Oceanogr. 56, 431–439 (2011).

    Article  Google Scholar 

  105. Jaspers, C., Møller, L. F. & Kiørboe, T. Salinity gradient of the Baltic Sea limits the reproduction and population expansion of the newly invaded comb jelly Mnemiopsis leidyi. PLoS ONE 6, e24065 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  106. Jaspers, C., Møller, L. F. & Kiørboe, T. Reproduction rates under variable food conditions and starvation in Mnemiopsis leidyi: significance for the invasion success of a ctenophore. J. Plankton Res. 37, 1011–1018 (2015).

    Article  Google Scholar 

  107. Jaspers, C., Marty, L. & Kiørboe, T. Selection for life-history traits to maximize population growth in an invasive marine species. Glob. Chang. Biol. 24, 1164–1174 (2018).

    PubMed  Article  Google Scholar 

  108. Reeve, M. R., Syms, M. A. & Kremer, P. Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency. J. Plankton Res. 11, 535–552 (1989).

    Article  Google Scholar 

  109. Jaspers, C. et al. Resilience in moving water: effects of turbulence on the predatory impact of the lobate ctenophore Mnemiopsis leidyi. Limnol. Oceanogr. 63, 445–458 (2018).

    Article  Google Scholar 

  110. Jaspers, C., Costello, J. H. & Colin, S. P. Carbon content of Mnemiopsis leidyi eggs and specific egg production rates in northern Europe. J. Plankton Res. 37, 11–15 (2015).

    CAS  Article  Google Scholar 

  111. Winnikoff, J. R., Haddock, S. H. D. & Budin, I. Depth- and temperature-specific fatty acid adaptations in ctenophores from extreme habitats. J. Exp. Biol. jeb.242800 (2021).

  112. Jaspers, C. et al. Microbiota differences of the comb jelly Mnemiopsis leidyi in native and invasive sub-populations. Front. Mar. Sci. 6, 635 (2019).

    Article  Google Scholar 

  113. Sutherland, K. R., Costello, J. H., Colin, S. P. & Dabiri, J. O. Ambient fluid motions influence swimming and feeding by the ctenophore Mnemiopsis leidyi. J. Plankton Res. 36, 1310–1322 (2014).

    Article  Google Scholar 

  114. Colin, S. P. et al. Elevating the predatory effect: sensory-scanning foraging strategy by the lobate ctenophore Mnemiopsis leidyi. Limnol. Oceanogr. 60, 100–109 (2015).

    Article  Google Scholar 

  115. Parker, G. H. The movements of the swimming-plates in ctenophores, with reference to the theories of ciliary metachronism. J. Exp. Zool. 2, 407–423 (1905).

    Article  Google Scholar 

  116. Baker, L. D. & Reeve, M. R. Laboratory culture of the lobate ctenophore Mnemiopsis mccradyi with notes on feeding and fecundity. Mar. Biol. 26, 57–62 (1974).

    Article  Google Scholar 

  117. Reeve, M. R., Walter, M. A. & Ikeda, T. Laboratory studies of ingestion and food utilization in lobate and tentaculate ctenophores. Limnol. Oceanogr. 23, 740–751 (1978).

    Article  Google Scholar 

  118. Swanberg, N. The feeding behavior of Beroe ovata. Mar. Biol. 24, 69–76 (1974).

    Article  Google Scholar 

  119. Haddock, S. H. D. Comparative feeding behavior of planktonic ctenophores. Integr. Comp. Biol. 47, 847–853 (2007).

    PubMed  Article  Google Scholar 

  120. Mayer, A. G. Ctenophores of the Atlantic Coast of North America (Carnegie Institution of Washington, 1912).

  121. Seravin, L. N. The systematic revision of the genus Mnemiopsis (Ctenophora, Lobata). 2. Species attribution of Mnemiopsis from the Black Sea and the species composition of the genus Mnemiopsis. Zool. Zh. 73, 19–34 (1994).

    Google Scholar 

  122. Bayha, K. M. et al. Worldwide phylogeography of the invasive ctenophore Mnemiopsis leidyi (Ctenophora) based on nuclear and mitochondrial DNA data. Biol. Invasions 17, 827–850 (2015).

    Article  Google Scholar 

  123. Costello, J. H., Sullivan, B. K., Gifford, D. J., Van Keuren, D. & Sullivan, L. J. Seasonal refugia, shoreward thermal amplification, and metapopulation dynamics of the ctenophore Mnemiopsis leidyi in Narragansett Bay, Rhode Island. Limnol. Oceanogr. 51, 1819–1831 (2006).

    Article  Google Scholar 

  124. Pang, K. & Martindale, M. Q. Ctenophore whole-mount in situ hybridization. CSH Protoc. 2008, db.prot5087 (2008).

  125. Salinas-Saavedra, M. & Martindale, M. Q. Improved protocol for spawning and immunostaining embryos and juvenile stages of the ctenophore Mnemiopsis leidyi. Protoc. Exchange https://doi.org/10.1038/protex.2018.092 (2018).

  126. Dieter, A. C., Vandepas, L. E. & Browne, W. E. in Whole-Body Regeneration: Methods and Protocols (eds. Blanchoud, S. & Galliot, B.) (Springer, 2022).

  127. Yamada, A., Martindale, M. Q., Fukui, A. & Tochinai, S. Highly conserved functions of the Brachyury gene on morphogenetic movements: insight from the early-diverging phylum Ctenophora. Dev. Biol. 339, 212–222 (2010).

    CAS  PubMed  Article  Google Scholar 

  128. Moreland, R. T. et al. A customized Web portal for the genome of the ctenophore Mnemiopsis leidyi. BMC Genomics 15, 316 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  129. Moreland, R. T., Nguyen, A.-D., Ryan, J. F. & Baxevanis, A. D. The Mnemiopsis Genome Project Portal: integrating new gene expression resources and improving data visualization. Database 2020, baaa029 (2020).

  130. Davidson, P. L. et al. The maternal–zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles. Mol. Reprod. Dev. 84, 1218–1229 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  131. Sebé-Pedrós, A. et al. Early metazoan cell type diversity and the evolution of multicellular gene regulation. Nat. Ecol. Evol. 2, 1176–1188 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  132. Levin, M. et al. The mid-developmental transition and the evolution of animal body plans. Nature 531, 637–641 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  133. Sachkova, M. Y. et al. Neuropeptide repertoire and 3D anatomy of the ctenophore nervous system. Curr. Biol. https://doi.org/10.1016/j.cub.2021.09.005 (2021).

    Article  PubMed  Google Scholar 

  134. Fidler, A. L. et al. Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues. eLife 6, (2017).

  135. Draper, G. W., Shoemark, D. K. & Adams, J. C. Modelling the early evolution of extracellular matrix from modern ctenophores and sponges. Essays Biochem. 63, 389–405 (2019).

    CAS  PubMed  Article  Google Scholar 

  136. Ryan, J. F. et al. The homeodomain complement of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa. Evodevo 1, 9 (2010).

    PubMed  PubMed Central  Article  Google Scholar 

  137. Maxwell, E. K., Ryan, J. F., Schnitzler, C. E., Browne, W. E. & Baxevanis, A. D. MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi. BMC Genomics 13, 714 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  138. Traylor-Knowles, N., Vandepas, L. E. & Browne, W. E. Still enigmatic: innate immunity in the ctenophore Mnemiopsis leidyi. Integr. Comp. Biol. 59, 811–818 (2019).

    CAS  PubMed  Article  Google Scholar 

  139. Felsenstein, J. Phylogenies and the comparative method. Am. Nat. 125, 1–15 (1985).

    Article  Google Scholar 

  140. Dunn, C. W., Giribet, G., Edgecombe, G. D. & Hejnol, A. Animal phylogeny and its evolutionary implications. Annu. Rev. Ecol. Evol. Syst. 45, 371–395 (2014).

    Article  Google Scholar 

  141. Giribet, G. Morphology should not be forgotten in the era of genomics—a phylogenetic perspective. Zool. Anz. 256, 96–103 (2015).

    Article  Google Scholar 

  142. Patry, W. L., Bubel, M., Hansen, C. & Knowles, T. Diffusion tubes: a method for the mass culture of ctenophores and other pelagic marine invertebrates. PeerJ 8, e8938 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  143. Baker, L. D. The Ecology of the Ctenophore Mnemiopsis mccradyi Mayer, in Biscayne Bay, Florida (Rosenstiel School of Marine and Atmospheric Science, 1973).

  144. Raskoff, K. A., Sommer, F. A., Hamner, W. M. & Cross, K. M. Collection and culture techniques for gelatinous zooplankton. Biol. Bull. 204, 68–80 (2003).

    PubMed  Article  Google Scholar 

  145. Greve, W. The “planktonkreisel”, a new device for culturing zooplankton. Mar. Biol. 1, 201–203 (1968).

    Article  Google Scholar 

  146. Ward, W. W. Aquarium systems for the maintenance of ctenophores and jellyfish and for the hatching and harvesting of brine shrimp (Artemia salina) larvae. Chesap. Sci. 15, 116–118 (1974).

    Article  Google Scholar 

  147. Kremer, P. Effect of food availability on the metabolism of the ctenophore Mnemiopsis mccradyi. Mar. Biol. 71, 149–156 (1982).

    Article  Google Scholar 

  148. Kremer, P. & Reeve, M. R. Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. II. Carbon budgets and growth model. J. Plankton Res. 11, 553–574 (1989).

    Article  Google Scholar 

  149. Harbison, G. R., Biggs, D. C. & Madin, L. P. The associations of Amphipoda Hyperiidea with gelatinous zooplankton—II. Associations with Cnidaria. Ctenophora Radiolaria. Deep Sea Res. I 24, 465–488 (1977).

    Article  Google Scholar 

  150. Laval, P. Hyperiid amphipods as crustacean parasitoids associated with gelatinous zooplankton. Oceanogr. Mar. Biol. Annu. Rev. 18, 11–56 (1980).

    Google Scholar 

  151. Yip, S. Y. Parasites of Pleurobrachia pileus Müller, 1776 (Ctenophora), from Galway Bay, western Ireland. J. Plankton Res. 6, 107–121 (1984).

    Article  Google Scholar 

  152. Martorelli, S. R. Digenea parasites of jellyfish and ctenophores of the southern Atlantic. Hydrobiologia 451, 305–310 (2001).

    Article  Google Scholar 

  153. Moss, A. G., Estes, A. M., Muellner, L. A. & Morgan, D. D. Protistan epibionts of the ctenophore Mnemiopsis mccradyi Mayer. Hydrobiologia 451, 295–304 (2001).

    Article  Google Scholar 

  154. Reitzel, A. M. et al. Ecological and developmental dynamics of a host–parasite system involving a sea anemone and two ctenophores. J. Parasitol. 93, 1392–1402 (2007).

    PubMed  Article  Google Scholar 

  155. Zeidler, W. & Browne, W. E. A new Glossocephalus (Crustacea: Amphipoda: Hyperiidea: Oxycephalidae) from deep-water in the Monterey Bay region, California, USA, with an overview of the genus. Zootaxa 4027, 408–424 (2015).

    PubMed  Article  Google Scholar 

  156. Reitzel, A. M., Daly, M., Sullivan, J. C. & Finnerty, J. R. Comparative anatomy and histology of developmental and parasitic stages in the life cycle of the lined sea anemone Edwardsiella lineata. J. Parasitol. 95, 100–112 (2009).

    PubMed  Article  Google Scholar 

  157. Pang, K. & Martindale, M. Q. Mnemiopsis leidyi spawning and embryo collection. CSH Protoc. 2008, db.prot5085 (2008).

    Google Scholar 

  158. Freeman, G. & Reynolds, G. T. The development of bioluminescence in the ctenophore Mnemiopsis leidyi. Dev. Biol. 31, 61–100 (1973).

    CAS  PubMed  Article  Google Scholar 

  159. Martindale, M. Q. & Henry, J. J. Experimental analysis of tentacle formation in the ctenophore Mnemiopsis leidyi. Biol. Bull. 193, 245–247 (1997).

    CAS  PubMed  Article  Google Scholar 

  160. Noda, N. & Tamm, S. L. Lithocytes are transported along the ciliary surface to build the statolith of ctenophores. Curr. Biol. 24, R951–R952 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  161. Tamm, S. L. & Moss, A. G. Unilateral ciliary reversal and motor responses during prey capture by the ctenophore Pleurobrachia. J. Exp. Biol. 114, 443–461 (1985).

    CAS  PubMed  Article  Google Scholar 

  162. Salinas-Saavedra, M. & Martindale, M. Q. Par protein localization during the early development of Mnemiopsis leidyi suggests different modes of epithelial organization in the metazoa. eLife 9, (2020).

  163. Technau, U. Brachyury, the blastopore and the evolution of the mesoderm. Bioessays 23, 788–794 (2001).

    CAS  PubMed  Article  Google Scholar 

  164. Papaioannou, V. E. The T-box gene family: emerging roles in development, stem cells and cancer. Development 141, 3819–3833 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  165. Yasuoka, Y., Shinzato, C. & Satoh, N. The mesoderm-forming gene brachyury regulates ectoderm-endoderm demarcation in the coral Acropora digitifera. Curr. Biol. 26, 2885–2892 (2016).

    CAS  PubMed  Article  Google Scholar 

  166. Servetnick, M. D. et al. Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral–aboral patterning. Development 144, 2951–2960 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  167. Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  168. Xiao, A. et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30, 1180–1182 (2014).

    CAS  PubMed  Article  Google Scholar 

  169. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  170. Gagnon, J. A. et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS ONE 9, e98186 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  171. Kistler, K. E., Vosshall, L. B. & Matthews, B. J. Genome engineering with CRISPR–Cas9 in the mosquito Aedes aegypti. Cell Rep. 11, 51–60 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  172. Varshney, G. K. et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res. 25, 1030–1042 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  173. Schultz, D. T. et al. A chromosome-scale genome assembly and karyotype of the ctenophore Hormiphora californensis. G3 https://doi.org/10.1093/g3journal/jkab302 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the anonymous reviewers for their time and generous feedback.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization: husbandry, W.P. and W.E.B.; genome editing, W.E.B. Methodology: husbandry, W.P. and W.E.B.; genome editing, J.S.P. and W.E.B. Investigation: husbandry, all authors; genome editing, J.S.P. and W.E.B. Validation: husbandry, all authors; genome editing, J.S.P. and W.E.B. Visualization: J.S.P. and W.E.B. Resources: husbandry, W.P. and W.E.B.; genome editing, W.E.B. Writing original draft: J.S.P., W.P. and W.E.B. Writing review and editing: all authors. Supervision: W.P. and W.E.B. Project administration: husbandry, W.P. and W.E.B.; genome editing, W.E.B. Funding acquisition: husbandry, W.P. and W.E.B.; genome editing, W.E.B.

Corresponding author

Correspondence to W. E. Browne.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Presnell, J. S. et al. Curr. Biol. 26, 2814–2820 (2016): https://doi.org/10.1016/j.cub.2016.08.019

Bessho-Uehara, M. et al. iScience 23, 101859 (2020): https://doi.org/10.1016/j.isci.2020.101859

Presnell, J. S. & Browne, W. E. Development 148, dev199771 (2021): https://doi.org/10.1242/dev.199771

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2

Reporting Summary

Supplementary Video 1

Demonstration of target feeding a Mnemiopsis leidyi adult. Zebrafish larvae (Danio rerio) that have been pre-washed with ASW are delivered directly to the tentacle lined feeding grooves via plastic pipette where they become ensnared in adhesive material produced by colloblast cells lining the tentilla. The trapped fish larvae are then transported orally, and engulfed

Supplementary Video 2

Juvenile Mnemiopsis leidyi cydippid capturing prey in tentacles. During juvenile stages, the two main tentacles extend into the surrounding water column from which side branching tentilla are then deployed to form a dense network of sticky colloblast cells used to ensnare nearby plankton

Supplementary Video 3

Juvenile Mnemiopsis leidyi cydippid characteristic tentacle deployment behavior. Cydippids typically swim a looping pattern, saturating the local water column with colloblast lined tentacles and tentilla to maximize prey capture

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Presnell, J.S., Bubel, M., Knowles, T. et al. Multigenerational laboratory culture of pelagic ctenophores and CRISPR–Cas9 genome editing in the lobate Mnemiopsis leidyi. Nat Protoc (2022). https://doi.org/10.1038/s41596-022-00702-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41596-022-00702-w

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing