Stable isotope-labelled prey
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| Bacterivory and prey identity (stable isotope probing) |
|---|
| Approach: RNA/DNA-SIP with 13C/15N-labelled prey; ultracentrifugation and amplicon sequencing of heavy fraction |
| Context: incubation, field |
| Spatial scale: point sample |
| Temporal scale: 24 h |
| Units: actively feeding taxa (presence/absence; relative enrichment) |
| Community captured: taxon-specific consumers identified by amplicon sequencing |
| Co-measurements: cellular abundances |
Method Overview
Bacterial or algal prey are grown on 13C- and/or 15N-labelled substrates, producing cells whose nucleic acids carry a heavy isotope label. These labelled prey are added to natural seawater samples and allowed to be consumed by community members over ~24 h. After incubation, community RNA or DNA is extracted and separated by isopycnic ultracentrifugation into light (unlabelled) and heavy (isotopically enriched, i.e., from organisms that consumed labelled prey) fractions[1]. Amplicon sequencing (16S, 18S, or ITS) of the heavy fraction identifies which community members consumed the labelled prey, revealing the taxonomic identity of active predators without prior culture.
Scale of measurement
The method provides qualitative and semi-quantitative information on which taxa are active predators under the incubation conditions. Incubations typically run 24 hours.
Data generated
Identities of actively feeding taxa (from 16S/18S amplicon sequencing of heavy RNA/DNA fraction). With quantitative SIP modifications, relative enrichment levels can be used to rank predator activity.
Units & currency
Units are actively feeding taxa (presence/relative abundance). The currency is actively feeding taxa identified by sequencing.
Sample size
Typical samples are ~1 L in volume.
Repositories & databases
Limitations
Indirect routes of label incorporation — such as excretion and re-uptake of labelled compounds, or transfer of label through multiple trophic levels — can lead to false-positive identification of non-grazing organisms in the heavy fraction. Sufficient incubation time is required for detectable label incorporation, but this increases the risk of indirect labelling. The method identifies active grazers but does not directly quantify per-cell ingestion rates.
Example Applications & Protocols
Classic examples
- Whiteley et al. (2007) RNA stable-isotope probing [1]
Recent applications
- Wilken et al. (2023) Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus [2]
- Orsi et al. (2018) Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing [3]
Common calculations/conversions
- Semi-quantitative enrichment: compare 16S/18S amplicon read relative abundance in heavy vs. light fraction to rank predator activity.
References
- ↑ 1.0 1.1 Whiteley, A. S., Thomson, B., Lueders, T., & Manefield, M. (2007). RNA stable-isotope probing. Nature Protocols, 2(4), 838–844. https://doi.org/10.1038/nprot.2007.115
- ↑ Wilken, S., Yung, C. C. M., Hamilton, M., Hoadley, K., Nzongo, J., Eckmann, C., Corrochano-Luque, M., Poirier, C., & Worden, A. Z. (2023). Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus. Proceedings of the National Academy of Sciences, 120(27), e2302388120. https://doi.org/10.1073/pnas.2302388120
- ↑ Orsi, W. D., Wilken, S., del Campo, J., Heger, T., James, E., Richards, T. A., Keeling, P. J., Worden, A. Z., & Santoro, A. E. (2018). Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing. Environmental Microbiology, 20(2), 815–827. https://doi.org/10.1111/1462-2920.14018