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Choice of methodology and surrogate prey are decisive for the quality of protistan bacterivory rate estimates
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.ORCID iD: 0000-0002-3284-3702
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden.ORCID iD: 0000-0002-4265-1835
2023 (English)In: Aquatic Microbial Ecology, ISSN 0948-3055, E-ISSN 1616-1564, Vol. 89, p. 43-53Article in journal (Refereed) Published
Abstract [en]

ABSTRACT: Microeukaryote predation on bacteria is a fundamental phenomenon to understand energy and nutrient dynamics at the base of the aquatic food web. To date, the most prevalent way to estimate grazing rates is by using epifluorescence microscopy to enumerate ingestion events of fluorescently labelled tracers (FLTs) after short-term incubation experiments. However, this approach can be sensitive to the type of FLT, requires skillful preparation of the samples and is limited to small sample sizes. We tested the susceptibility of rate estimates to the choice of prey and made a side-by-side comparison between microscopy and flow cytometry when recording ingestion by a bacterivorous flagellate. Short-term uptake experiments were established using 5 types of FLTs differing in quality (living, dead or inert) and size (large or small), with <i>Ochromonas triangulata</i> as a model flagellate. The experiments showed that (1) each of the different prey types yielded different clearing rates, ranging from 0.5 to 3.6 nl cell<sup>-1</sup> h<sup>-1</sup>, with the largest differences (3-fold or higher) between small prey (lower rates) and large prey (higher rates); (2) the cytometry estimate differed significantly from the microscopy estimate in 3 out of 4 experimental configurations; and (3) the precision of the cytometric analysis was greater, with >3-fold higher uncertainty associated with microscopy counting. Our results validate that flow cytometry provides a more precise bacterivory estimate, and that the choice of FLT influences the grazing rate estimate to a high extent regardless of the analytical method used.

Place, publisher, year, edition, pages
Inter-Research Science Publisher , 2023. Vol. 89, p. 43-53
Keywords [en]
Bacterivory rates, Fluorescently labelled tracers, FLB, Flow cytometry, Ochromonas triangulata
National Category
Ecology Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-500711DOI: 10.3354/ame01996ISI: 000989677200001OAI: oai:DiVA.org:uu-500711DiVA, id: diva2:1752419
Available from: 2023-04-21 Created: 2023-04-21 Last updated: 2023-06-28Bibliographically approved
In thesis
1. Single-cell methodologies for ecological and metabolic mapping of mixotrophic microeukaryotes
Open this publication in new window or tab >>Single-cell methodologies for ecological and metabolic mapping of mixotrophic microeukaryotes
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mixotrophy in aquatic protists is pivotal for our understanding of aquatic microbial food web dynamics. This thesis is centered around aquatic unicellular mixotrophs, and comprises three methodological approaches aimed to tackle mixotroph ecology at single-cell resolution: the identification of actively feeding mixotrophs in natural samples, the determination of specific interactions among mixotrophs and bacterial prey, and the profiling of two distinct mixotrophic populations based on the gene expression of their constitutive individuals.

First, we investigated the feasibility of cytometrically sorting actively feeding mixotrophs from a natural community. The approach was based on the use of fluorescently labelled feeding tracers (FLTs) in conjunction with chloroplast autofluorescence from the feeding cell to retrieve mixotrophic individuals for subsequent single cell characterization by sequencing of a taxonomic marker gene. The preference for different FLT types showed that for mixotrophs in culture, FLT size was the strongest factor influencing FLT-based capture. This approach was then used to identify actively feeding mixotrophs from a lake water sample. The method proved to be both highly selective and specific and allowed the identification of an active natural mixotrophic community of unexpected diversity.

Secondly, we explored the potential of adapting emulsion, paired-isolation and concatenation PCR (epicPCR) to uncover physical connections between individual unicellular eukaryotes and their associated bacterial cohort. The results from three proof-of-concept experiments, however, did not conform to the expectations and showcased several deficiencies that need to be addressed. Mainly, the frequency of recovered links showed that the protocol, as deployed in our experiments, was prone to yield spurious abundance-driven associations between the eukaryotes and bacteria, since the most abundant bacteria were the ones driving the strongest associations with our test predators. Nevertheless, we identify possible solutions and point to avenues for future development to overcome the current limitations.

Finally, the capability of full-transcript single-cell RNA sequencing was surveyed to provide a reliable transcriptomic landscape of a non-mammalian, non-model eukaryotic organism with no available reference genome. We could show that, while some of the detailed functional information might remain uncharacterized, the workflow provide sufficient raw data to resolve population structure based on expression profiles.

In summary, with varying degrees of success, these attempts to expose and study mixotrophic unicellular eukaryotes demonstrate that the time is ripe to explore the ecology of mixotrophs at single-cell level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2023. p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2285
Keywords
mixotrophy, single-cell
National Category
Ecology Microbiology
Identifiers
urn:nbn:se:uu:diva-506111 (URN)978-91-513-1847-9 (ISBN)
Public defence
2023-09-22, Friessalen, Evolutionsbiologiskt centrum, Norbyvägen 14, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2023-08-31 Created: 2023-06-28 Last updated: 2023-08-31

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Florenza, JavierBertilsson, Stefan

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