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

Nature Protocols volume 17, pages 1868–1900 (2022)Cite this article

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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.

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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.

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Data availability

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

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Acknowledgements

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

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Author notes

  1. J. S. Presnell

    Present address: Department of Human Genetics, University of Utah, Salt Lake City, UT, USA

  2. These authors contributed equally: J. S. Presnell, W. E. Browne.

Authors and Affiliations

  1. Department of Biology, University of Miami, Miami, FL, USA

    J. S. Presnell & W. E. Browne

  2. Monterey Bay Aquarium, Monterey, CA, USA

    M. Bubel, T. Knowles & W. Patry

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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.

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Correspondence to W. E. Browne.

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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

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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 17, 1868–1900 (2022). https://doi.org/10.1038/s41596-022-00702-w

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