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Experimental manipulation of microbiota reduces host thermal tolerance and fitness under heat stress in a vertebrate ectotherm

Nature Ecology & Evolution volume 6, pages 405–417 (2022)Cite this article

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Abstract

Identifying factors that influence how ectothermic animals respond physiologically to changing temperatures is of high importance given current threats of global climate change. Host-associated microbial communities impact animal physiology and have been shown to influence host thermal tolerance in invertebrate systems. However, the role of commensal microbiota in the thermal tolerance of ectothermic vertebrates is unknown. Here we show that experimentally manipulating the tadpole microbiome through environmental water sterilization reduces the host’s acute thermal tolerance to both heat and cold, alters the thermal sensitivity of locomotor performance, and reduces animal survival under prolonged heat stress. We show that these tadpoles have reduced activities of mitochondrial enzymes and altered metabolic rates compared with tadpoles colonized with unmanipulated microbiota, which could underlie differences in thermal phenotypes. These results demonstrate a strong link between the microbiota of an ectothermic vertebrate and the host’s thermal tolerance, performance and fitness. It may therefore be important to consider host-associated microbial communities when predicting species’ responses to climate change.

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Fig. 1: Effects of microbial colonization treatment and acclimation temperature on tadpole gut microbial communities, morphometrics and acute thermal tolerance.
Fig. 2: Survival of colonized and depleted tadpoles under heat stress conditions.
Fig. 3: Maximum swimming velocity of colonized and depleted tadpoles at six assay temperatures.
Fig. 4: Mitochondrial enzyme activities in tail muscle of colonized and depleted tadpoles at three assay temperatures.
Fig. 5: Relationship between mass-specific resting metabolic rate and body mass in colonized and depleted tadpoles at two temperatures.

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Data availability

The raw microbiome sequencing data are available from NCBI’s Sequence Read Archive under accession no. PRJNA732310. All other raw datasets are available from the Zenodo repository at https://doi.org/10.5281/zenodo.5703371.

Code availability

The R code used for the statistical analyses in this study is available from the Zenodo repository at https://doi.org/10.5281/zenodo.5703371.

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Acknowledgements

We thank M. Ohmer, K. Altman and E. Le Sage for field collection assistance; K. Kohler, M. Maurer, M. Maier, S. Reilly, A. Haid, C. Duckworth and J. Adams for animal husbandry and DNA extraction assistance; and N. Barts for technical assistance. We also thank the University of Pittsburgh’s Health Sciences Metabolomics and Lipidomics core facility (NIH S10OD023402 PI Wendell), the DNA Services Facility at the University of Illinois at Chicago, and the Microbial Analysis, Resources, and Services Facility at the University of Connecticut for sample processing. This work was supported by the University of Pittsburgh (start-up funds to K.D.K.), Elmhurst University (faculty research grant to P.M.M.) and the National Science Foundation (GRFP to S.S.F.).

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Authors and Affiliations

  1. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA

    Samantha S. Fontaine & Kevin D. Kohl

  2. Department of Biology, Elmhurst University, Elmhurst, IL, USA

    Patrick M. Mineo

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  1. Samantha S. Fontaine
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Contributions

S.S.F., K.D.K. and P.M.M. designed the study. S.S.F. and P.M.M. collected the data. S.S.F. analysed the data, generated the figures and wrote the initial manuscript draft with editing from K.D.K. and P.M.M. K.D.K. supervised the research.

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Correspondence to Samantha S. Fontaine.

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The authors declare no competing interests.

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Nature Ecology & Evolution thanks Camila Carlos-Shanley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Experimental designs and sample sizes for experiments 1 and 2.

For both experiments, tadpoles were reared from pairs of adult frogs in the laboratory and divided into colonized (C) and depleted (D) water treatments at Gosner stage (GS) 25. For experiment 1, green frog tadpoles and pond water from Louisiana (LA, USA) were used and for experiment 2, green frog tadpoles and pond water from Pennsylvania (PA, USA) were used. Images created with BioRender.com.

Extended Data Fig. 2 Environmental microbial communities of pond water used for the colonized microbial treatment.

a, Principal Coordinate (PCo) analysis plot based on Bray-Curtis dissimilarity between microbial communities of water samples collected fresh from the pond or after storage in the laboratory at 4 °C. b, Principal Coordinate (PCo) analysis plot based on Bray-Curtis dissimilarity between tadpole gut microbial communities and microbial communities of pond water collected fresh from the pond or after storage in the laboratory. Due to overlap between water types on the plot, fresh pond water samples are outlined in black. For both PCoA plots, percentages represent the proportion of variation explained by each axis. c, mean relative abundances of bacterial phyla found in pond water samples fresh from the pond or after storage in the laboratory. The top ten most abundant phyla are shown individually, and the remainder are grouped together as “other”. Any bacteria that were unable to be assigned to a phylum are grouped together as “unassigned”. d, mean relative abundances of bacterial genera found in pond water samples fresh from the pond or after storage in the laboratory. The top ten most abundant genera from each group are shown individually, and the remainder are grouped together as “other”. Any bacteria that were unable to be assigned to a genus are grouped together as “unassigned”. For all figures, N = 2 for fresh pond samples and N = 7 for stored samples.

Extended Data Fig. 3 Mean relative abundances of bacterial phyla in gut microbial communities of tadpoles from experiment 1 across microbial colonization and acclimation temperature treatment groups.

The top ten most abundant phyla are shown individually, and the remainder are grouped together as “other”. Any bacteria that were unable to be assigned to a phylum are grouped together as “unassigned”. N = 27 animals per group.

Extended Data Fig. 4 Impacts of microbial colonization treatment and acclimation temperature on tadpole gut microbial communities in experiment 1.

a, Faith’s phylogenetic diversity of the gut bacterial community b, Shannon diversity of the gut bacterial community c, Pielou’s evenness within the gut bacterial community d, Number of bacterial cells in tadpole gut contents measured using flow cytometry and shown on a log scale e, Principal Coordinate (PCo) analysis plot based on Unweighted UniFrac distance between gut bacterial community samples f, Principal Coordinate analysis (PCo) plot based on Weighted UniFrac distance between gut bacterial community samples. For boxplots a-c, N = 25 for the 14 °C colonized and depleted groups, 26 for the 22 °C colonized and depleted groups, 26 for the 28 °C depleted group, and 27 for the 28 °C colonized group. For boxplot d, N = 3 for the 14 °C colonized group, 10 for the 14 °C depleted group, 10 for the 22 °C colonized group, 11 for the 22 °C depleted group, 10 for the 28 °C depleted group, and 9 for the 28 °C colonized group. For all boxplots, the center line represents the median, the length of the box extends through the IQR, and whiskers extend to 1.5x IQR. All points outside this range are plotted individually. For all principal coordinate analysis plots, percentages represent the proportion of variation explained by each axis. C = colonized tadpoles and D = depleted tadpoles. Colors represent tadpole acclimation temperature.

Extended Data Fig. 5 Plots used to determine gating parameters for flow cytometry from one representative sample.

a, A plot of fluorescein isothiocyanate (FITC) vs. cell counts was used to distinguish cells stained with SYBR Green dye from all other cells b, A plot of forward scatter (FSC) vs. side scatter (SSC) was used to distinguish populations of counting beads from all other events. To establish initial gates for counting beads, blank samples spiked only with beads were used. On both plots, red rectangles represent the gates and surround the events counted. Percentages indicate the proportion of events within the gate out of total events. R1 = stained bacterial cells and R2 = counting beads.

Extended Data Fig. 6 Tadpole morphometrics across microbial colonization and acclimation temperature treatment groups from experiment 1.

a, tadpole body length b, tadpole body width c, tadpole facial symmetry, calculated as the absolute value, subtracted from 1, of the difference between the distance from the center of each eye to the tip of the nose. The center line of each boxplot represents the median, the length of the box extends through the IQR, and whiskers extend to 1.5x IQR. All points outside this range are plotted individually. C = colonized tadpoles and D = depleted tadpoles. Colors represent tadpole acclimation temperature. N = 27 animals per group.

Extended Data Fig. 7 Impact of microbial colonization treatment on tadpole gut microbial communities in experiment 2.

a, Number of bacterial ASVs in the tadpole gut bacterial community, b, Faith’s phylogenetic diversity of the tadpole gut bacterial community, c, Pielou’s evenness of the tadpole gut bacterial community, and d, Principal Coordinate (PCo) analysis plot based on Bray-Curtis dissimilarity between gut bacterial community samples. Percentages represent the proportion of variation explained by each axis. The center line of each boxplot represents the median, the length of the box extends through the IQR, and whiskers extend to 1.5x IQR. All points outside this range are plotted individually. C = colonized tadpoles and D = depleted tadpoles. N = 13 colonized animals and 15 depleted animals.

Extended Data Fig. 8 The impact of microbial colonization treatment on tadpole morphometrics and heat tolerance in experiment 2.

a, tadpole body mass b, tadpole body length c, tadpole developmental stage based on the Gosner system d, tadpole critical thermal maximum (CTmax). The center line of each boxplot represents the median, the length of the box extends through the IQR, and whiskers extend to 1.5x IQR. All points outside this range are plotted individually. C = colonized tadpoles and D = depleted tadpoles. For boxplots a and b, N = 19 colonized animals and 21 depleted animals. For boxplot c, N = 19 colonized animals and 20 depleted animals. For boxplot d, N = 17 colonized animals and 18 depleted animals.

Extended Data Fig. 9 Impacts of microbial colonization treatment and assay temperature on tadpole mass-specific resting metabolic rate.

The center line of each boxplot represents the median, the length of the box extends through the IQR, and whiskers extend to 1.5x IQR. All points outside this range are plotted individually. C = colonized tadpoles and D = depleted tadpoles. On the y-axis VO2 refers to oxygen consumption. N = 10 colonized animals per temperature and 9 depleted animals per temperature.

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Fontaine, S.S., Mineo, P.M. & Kohl, K.D. Experimental manipulation of microbiota reduces host thermal tolerance and fitness under heat stress in a vertebrate ectotherm. Nat Ecol Evol 6, 405–417 (2022). https://doi.org/10.1038/s41559-022-01686-2

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