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Abstract The reef-building corals can thrive in nutrient-poor waters because of the mutualistic symbiosis between the animal hosts and their photosynthetic dinoflagellate endosymbionts. This symbiosis is threatened by climate change and other anthropogenic stressors, so that a deeper mechanistic understanding of its function is not only of great basic biological interest but also crucial for developing rational approaches to coral conservation. The small sea anemone Aiptasia is an attractive model system for studies of this symbiosis but has been limited to date by a lack of effective genetic methods. Here, we describe the use of a simple electroporation protocol to introduce various genetic constructs [plasmid DNAs, mRNAs, and short-hairpin (sh) RNAs] into Aiptasia zygotes. Plasmid-based expression of reporter constructs in the resulting larvae was highly mosaic. In contrast, electroporation of mRNAs into zygotes resulted in uniform expression within the larvae, and success rates were similar when single or multiple mRNAs were introduced. The shRNAs were effective in knocking down expression of both coelectroporated mRNAs and endogenous genes. In this way, we could confirm the previously reported role of BRACHYURY in cnidarian embryonic development. In addition, we could show that knockdown of an Aiptasia homologue of the lysosomal-associated membrane protein 1 interfered with larval uptake and/or retention of a symbiosis-compatible algal strain. The ability to use Aiptasia larvae for such reverse-genetic studies should greatly enhance the power of this model system and serve as a starting point for further development of genetic tools in Aiptasia and other cnidarians.
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Applying model approaches in non-model systems: A review and case study on coral cell culture
Model systems approaches search for commonality in patterns underlying biological diversity and complexity led by common evolutionary paths. The success of the approach does not rest on the species chosen but on the scalability of the model and methods used to develop the model and engage research. Fine-tuning approaches to improve coral cell cultures will provide a robust platform for studying symbiosis breakdown, the calcification mechanism and its disruption, protein interactions, micronutrient transport/exchange, and the toxicity of nanoparticles, among other key biological aspects, with the added advantage of minimizing the ethical conundrum of repeated testing on ecologically threatened organisms. The work presented here aimed to lay the foundation towards development of effective methods to sort and culture reef-building coral cells with the ultimate goal of obtaining immortal cell lines for the study of bleaching, disease and toxicity at the cellular and polyp levels. To achieve this objective, the team conducted a thorough review and tested the available methods (i.e. cell dissociation, isolation, sorting, attachment and proliferation). The most effective and reproducible techniques were combined to consolidate culture methods and generate uncontaminated coral cell cultures for ~7 days (10 days maximum). The tests were conducted on scleractinian corals Pocillopora acuta of the same genotype to harmonize results and reduce variation linked to genetic diversity. The development of cell separation and identification methods in conjunction with further investigations into coral cell-type specific metabolic requirements will allow us to tailor growth media for optimized monocultures as a tool for studying essential reef-building coral traits such as symbiosis, wound healing and calcification at multiple scales.
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- PAR ID:
- 10287463
- Editor(s):
- Voolstra, Christian R.
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 16
- Issue:
- 4
- ISSN:
- 1932-6203
- Page Range / eLocation ID:
- e0248953
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0248953
