SupplementaryMaterial
Effects of B. cereusendospores on free-livingprotistgrowth
Susana S. Santos1, Niels Bohse Hendriksen1, Hans Henrik Jakobsen2, Anne Winding1#
1Department of Environmental Science, Aarhus University, Denmark
2Department of Bioscience,Aarhus University, Denmark
#correspondingauthor:
Anne Winding
Department of Environmental Science
University of Aarhus
Frederiksborgvej 399,
DK-4000 Roskilde, Denmark
Phone: +45 87158615
e-mail:
Table S1: Data for the nonlinear leastsquaresfitting model when data werefitted to the Gompertzsigmoidequation: f=a*exp(-exp(-(x-x0)/b)). Significant differences between the slopes, withineachprotiststrainwere not observed (ANOVA; P 0.05).
Predator / B. cereus stage / R2 / Asymptote (A) / p(A) / Slope (b) / p(b)(± SE) / (± SE)
T. pyriformis
(ATCC 30005) / Vegetative cells / 0.7 / 15.79 ± 1.01 / <0.0001 / 11.89 ± 3.77 / 0.0048
Endospores (Active) / 0.8 / 13.81 ± 0.91 / <0.0001 / 17.12 ± 5.56 / 0.0057
Endospores (Inactive) / 0.3 / 11.75 ± 37.62 / 0.75 / 0.99 ± 0.02 / 0.916
A. castellanii(ATCC 50373) / Vegetative cells / 0.8 / 13.38 ± 0.87 / <0.0001 / 20.56 ± 5.61 / 0.0015
Endospores (Active) / 0.7 / 14.63 ± 2.45 / <0.0001 / 36.38 ± 22.21 / 0.1163
Endospores (Inactive) / 0.5 / 9.39 ± 0.45 / <0.0001 / 3.24 ± 1.56 / 0.0507
Cercomonassp.
(ATCC 50334) / Vegetative cells / 0.7 / 13.48 ± 0.56 / <0.0001 / 7.33 ± 2.44 / 0.0068
Endospores (Active) / 0.6 / 9.84 ± 0.36 / <0.0001 / 2.28 ± 0.89 / 0.0192
Endospores (Inactive) / 0.7 / 11.71 ± 0.36 / <0.0001 / 2.36 ± 0.76 / 0.0056
Table S2: Data for the linear regression modelling for prediction of protistgrowth from ingestions rates (above ≈ 102preyparticles h-1 for T. pyriformisand Cercomonassp.). Significant differences between the slopeswere not observed (Fowler et al., 1998; P 0.05).
Predator / B. cereus stage / R2 / Slope (± SE) / p(b)T. pyriformis
(ATCC 30005) / Vegetative cells / 0.2 / 4.66E-08 ± 2.32E-06 / 0.985
Endospores (Active) / 0.2 / 2.67E-05 ± 3.75E-05 / 0.517
Endospores (Inactive) / 0.7 / 1.23E-06 ± 3.27E-07 / 0.013
A. castellanii
(ATCC 50373) / Vegetative cells / 0.2 / 4.37E-05 ± 4.75E-05 / 0.43
Endospores (Active) / 0.9 / 7.97E-05 ± 1.84E-05 / 0.049
Endospores (Inactive) / 0.6 / 5.32E-05 ± 1.83E-05 / 0.027
Cercomonassp.
(ATCC 50334) / Vegetative cells / 0.4 / 3.96E-04 ± 2.00E-04 / 0.095
Endospores (Active) / 0.6 / 4.71E-05 ± 1.54E-05 / 0.023
Endospores (Inactive) / 0.7 / 8.75E-05 ± 2.16E-05 / 0.007
Impact of pH on RedCellTrackerfluorescence
The First et al. (2012) protocolwasadapted tostudy the impact of pH on CTR-labelled B. cereusfluorescence. To test whetherintact or disruptedbacterialcellsremainfluorescent in acidicconditions, a pH range of 2,5 – 7wasestablished by addingNaOH or HCl to 10 mL AS buffer (Lekfeldt & Rønn, 2008). B. cereus vegetative cells and endosporeswere labelled with CTR as describedpreviously. Afterincubation, cellswerecentrifuged at 10000g for 5 min. The supernatantcontained CTR in solution wascollected and keptrefrigerated in the dark untilanalysis. The pelletedcellswerewashed 4 times and re-suspended in 1.5 mL of AS buffer to removeany traces of extracellular CTR. Fluorescence of cellswasmeasured by adding 20 µL of either the cell suspension (with intactcells), extracellular suspension and cell suspension after 20 min of sonication, to 180 µL of the different pH treatments. Triplicates of relative fluorescenceintensities analyses wereperformed in 96-wells plates in a PlateChameleonfluorescenceplatereader (Hidex, Finland).
Fig. S1: The effect of pH on the fluorescence of CTR-labelled B. cereusendospores, on extracellularfree CTR in solution and or on disruptB. cereuscells. Treatments with significantly differences within the same pH aredenoted with different letters. Treatments with significantlydifferentfluorescencecompared with pH 7 aredenoted by an asterisk. Errors bars = ± SE.
Fig. S2:Epifluorescence images of DAPI stained (blue) T. pyriformis (A-I) and A. castellanii (J-R) recovered from co-incubation with CTR-staining (red) B. cereusvegetative cells (A-C; J-L) activeendospores (D-F; M-O) and inactivatedendospores (G-I; P-Q). Bacteriaareintact and taken up in protistvacuoles.
Figure S3: Control cultures of B. cereuslife forms (A) and the differentprotiststrains (B) quantified by qPCR, targeting 16S and 18S rDNA genes, respectively. Resultscorrespond to the average of three independent experiments, and means and standard errorsareshown. Data does not differsignificantlyalong time (P > 0.05; Tukey). Errors bars = ± SE.
Fig. S4:Functionalresponses of diverse protistspredators to B. cereuslife forms. Lines depictfunctionalresponse model fits to the data. To test which type of functionalresponsecurvedescribed the relationshipbetweenpreyconsumption rate and preydensitybest, wefitted non-linear mixed effects models to eachprotist and B. cereuslife forms, separately. The type I (polynomial), II (similar to Holling’s disc equation) and III functionalresponse (sigmoid) wereused in the data fitting. R2 and F statisticwereused to test the fitness of the models. Analyses wereperformed with SigmaPlot (SigmaPlot 11.0 software, Erkrath, Germany).
Fig. S5:Comparison of T. pyriformismorphologywhen fed with differentqualityfood: B. cereus vegetative cells (A), Pseudomonaschlororaphis (ATCC43928) (B), B. cereusendospores (Active) (C) B. cereusendospores (Inactive) (D) and ATCC357 growth media (E).Notice the highaccumulation of large foodvacuoles in A and B.
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