BrdU-labeled prey incorporation into things with chloroplasts
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| Constitutive mixotrophy (BrdU-labelled prey) |
|---|
| Approach: BrdU-labelled bacteria ingested by pigmented cells; immunofluorescence detection |
| Context: field, lab |
| Spatial scale: point sample |
| Temporal scale: 24 h |
| Units: active mixotrophic cells L-1 |
| Community captured: constitutive mixotrophs (autofluorescent cells with ingested BrdU-labelled bacteria) |
| Co-measurements: protist abundance |
Method Overview
Bacteria are labelled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU), which is incorporated into newly synthesized DNA during active growth. The BrdU-labelled bacteria are added to seawater samples, which are incubated for ~24 h to allow ingestion and digestion. After fixation, cells are filtered onto polycarbonate membranes and probed with anti-BrdU antibodies conjugated to a fluorescent secondary label (e.g., Alexa Fluor). Cells that are simultaneously autofluorescent (indicating plastids) and BrdU-positive (indicating ingested labelled bacteria) are identified as constitutive mixotrophs engaging in phagotrophy. This approach directly detects bacterivory in phytoplankton without relying on proxy particles[1].
Scale of measurement
Incubation of 24 hours for each sample.
Data generated
Abundance of constitutively mixotrophic cells (cells L-1) and their proportion within the phytoplankton community. Potentially, the degree of activity (number of BrdU-positive inclusions per cell) can also be assessed.
Units & currency
Units are active mixotrophic cells L-1. The currency is number of active constitutive mixotrophs.
Sample size
Typical samples are ~1 L in volume.
Repositories & databases
Limitations
The method assumes that BrdU in prey nucleotides (not just surface-attached bacteria) is the source of BrdU signal in plastid-containing cells. Non-specific antibody binding or carry-over of BrdU-labelled fragments from lysed bacteria could produce false positives. The 24 h incubation may allow secondary incorporation of BrdU from labelled bacteria that have been grazed, lysed, and whose DNA fragments are re-assimilated.
Example Applications & Protocols
Classic examples
Recent applications
- Millette et al. (2023) Mixoplankton and mixotrophy: future research priorities [1]
Common calculations/conversions
- % constitutive mixotrophs = (BrdU+ and autofluorescent cells) / (total autofluorescent cells) × 100.
References
- ↑ 1.0 1.1 Millette, N. C., Leles, S. G., Johnson, M. D., & Menden-Deuer, S. (2023). Mixoplankton and mixotrophy: future research priorities. Journal of Plankton Research, 45(4), 576–596. https://doi.org/10.1093/plankt/fbad020