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Title: Multigenerational laboratory culture of pelagic ctenophores and CRISPR/Cas9 genome editing in the lobate, Mnemiopsis leidyi
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. more »« less
The ability to isolate, monitor, and examine specific cells of interest enables targeted experimental manipulations that would otherwise be difficult to perform and interpret in the context of the whole organism. In vitro primary cell cultures derived from ctenophores thus serve as an important tool for understanding complex cellular and molecular interactions that take place both within and between various ctenophore cell types. Here we describe methods for reliably generating and maintaining primary cell cultures derived from the lobate ctenophore Mnemiopsis leidyi that can be used for a wide variety of experimental applications.
Ctenophores (comb jellies) are emerging as important animals for investigating fundamental questions across numerous branches of biology (e.g., evodevo, neuroscience and biogeography). A few ctenophore species including, most notably,Mnemiopsis leidyi, are considered as invasive species, adding to the significance of studying ctenophore ecology. Despite the growing interest in ctenophore biology, relatively little is known about their reproduction. Like most ctenophores,M. leidyiis a simultaneous hermaphrodite capable of self-fertilization. In this study, we assess the influence of light on spawning, the effect of body size on spawning likelihood and reproductive output, and the cost of self-fertilization on egg viability inM. leidyi. Our results suggest thatM. leidyispawning is more strongly influenced by circadian rhythms than specific light cues and that body size significantly impacts spawning and reproductive output.Mnemiopsis leidyiadults that spawned alone produced a lower percentage of viable embryos versus those that spawned in pairs, suggesting that self-fertilization may be costly in this species. These results provide insight into the reproductive ecology ofM. leidyiand provide a fundamental resource for researchers working with them in the laboratory.
ABSTRACT Escherichia coli can survive for long periods in batch culture in the laboratory, where they experience a stressful and heterogeneous environment. During this incubation, E. coli acquires mutations that are selected in response to this environment, ultimately leading to evolved populations that are better adapted to these complex conditions, which can lead to a better understanding of evolutionary mechanisms. Mutations in regulatory genes often play a role in adapting to heterogeneous environments. To identify such mutations, we examined transcriptional differences during log phase growth in unaged cells compared to those that had been aged for 10 days and regrown. We identified expression changes in genes involved in motility and chemotaxis after adaptation to long-term cultures. We hypothesized that aged populations would also have phenotypic changes in motility and that motility may play a role in survival and adaptation to long-term cultures. While aged populations did show an increase in motility, this increase was not essential for survival in long-term cultures. We identified mutations in the regulatory gene sspA and other genes that may contribute to the observed differences in motility. Taken together, these data provide an overall picture of the role of mutations in regulatory genes for adaptation while underscoring that all changes that occur during evolution in stressful environments are not necessarily adaptive. IMPORTANCE Understanding how bacteria adapt in long-term cultures aids in both better treatment options for bacterial infections and gives insight into the mechanisms involved in bacterial evolution. In the past, it has been difficult to study these organisms in their natural environments. By using experimental evolution in heterogeneous and stressful laboratory conditions, we can more closely mimic natural environments and examine evolutionary mechanisms. One way to observe these mechanisms is to look at transcriptomic and genomic data from cells adapted to these complex conditions. Here, we found that although aged cells increase motility, this increase is not essential for survival in these conditions. These data emphasize that not all changes that occur due to evolutionary processes are adaptive, but these observations could still lead to hypotheses about the causative mutations. The information gained here allow us to make inferences about general mechanisms underlying phenotypic changes due to evolution.
Norekian, Tigran P.; Moroz, Leonid L.(
, Journal of Morphology)
Abstract
Ctenophores are descendants of an early branching basal metazoan lineage, which may have evolved neurons and muscles independently from other animals.Mnemiopsisis one of the important reference ctenophore species. However, little is known about its neuromuscular organization. Here, we mapped and tracked the development of the neural and muscular elements in the early hatching cydippid larvae, as well as adultMnemiopsis leidyi. The overall development of the neuromuscular system inMnemiopsiswas very similar toPleurobrachia bachei, although inMnemiopsisthe entire process occurred significantly faster. The subepithelial neural cells were observed immediately after hatching. This population consisted of a dozen of separated individual neurons with short neurites. In about 2 days, when their neurites grew significantly longer and connected to their neighbors, they began to form a canonical polygonal subepithelial network. Mesogleal neural elements prominent in all studied adult ctenophores were not detectable inMnemiopsislarvae but were clearly labeled in closely related Lobata speciesBolinopsis infundibulum. Hatched larvae also had putative mechanoreceptors with long stereocilia and approximately two dozen muscle cells. In adultMnemiopsis,the feeding lobes and auricles contained two distinct populations of neurons and neural ensembles that were not observed in other ctenophore lineages and likely represented elaborate neuronal innovations characteristic for the clade Lobata and their lifestyles.
Lynch, Michael; Wei, Wen; Ye, Zhiqiang; Pfrender, Michael(
, Proceedings of the National Academy of Sciences)
Despite evolutionary biology’s obsession with natural selection, few studies have evaluated multigenerational series of patterns of selection on a genome-wide scale in natural populations. Here, we report on a 10-y population-genomic survey of the microcrustaceanDaphnia pulex.The genome sequences of800 isolates provide insights into patterns of selection that cannot be obtained from long-term molecular-evolution studies, including the following: the pervasiveness of near quasi-neutrality across the genome (mean net selection coefficients near zero, but with significant temporal variance about the mean, and little evidence of positive covariance of selection across time intervals); the preponderance of weak positive selection operating on minor alleles; and a genome-wide distribution of numerous small linkage islands of observable selection influencing levels of nucleotide diversity. These results suggest that interannual fluctuating selection is a major determinant of standing levels of variation in natural populations, challenge the conventional paradigm for interpreting patterns of nucleotide diversity and divergence, and motivate the need for the further development of theoretical expressions for the interpretation of population-genomic data.
Presnell, J. S., Bubel, M., Knowles, T., Patry, W., and Browne, W. E. Multigenerational laboratory culture of pelagic ctenophores and CRISPR/Cas9 genome editing in the lobate, Mnemiopsis leidyi. Retrieved from https://par.nsf.gov/biblio/10348385. Nature protocols 17. Web. doi:10.1038/s41596-022-00702-w.
Presnell, J. S., Bubel, M., Knowles, T., Patry, W., & Browne, W. E. Multigenerational laboratory culture of pelagic ctenophores and CRISPR/Cas9 genome editing in the lobate, Mnemiopsis leidyi. Nature protocols, 17 (). Retrieved from https://par.nsf.gov/biblio/10348385. https://doi.org/10.1038/s41596-022-00702-w
Presnell, J. S., Bubel, M., Knowles, T., Patry, W., and Browne, W. E.
"Multigenerational laboratory culture of pelagic ctenophores and CRISPR/Cas9 genome editing in the lobate, Mnemiopsis leidyi". Nature protocols 17 (). Country unknown/Code not available. https://doi.org/10.1038/s41596-022-00702-w.https://par.nsf.gov/biblio/10348385.
@article{osti_10348385,
place = {Country unknown/Code not available},
title = {Multigenerational laboratory culture of pelagic ctenophores and CRISPR/Cas9 genome editing in the lobate, Mnemiopsis leidyi},
url = {https://par.nsf.gov/biblio/10348385},
DOI = {10.1038/s41596-022-00702-w},
abstractNote = {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.},
journal = {Nature protocols},
volume = {17},
author = {Presnell, J. S. and Bubel, M. and Knowles, T. and Patry, W. and Browne, W. E.},
}
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