Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria

Abstract

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.

Figure 1

Experimental design: a natural HNF community in 5-μm filtered water from Římov reservoir was pre-incubated for 30 h and then subjected to additions of different bacterial food items as the major HNF food source (for details see Methods). Note that the initial concentration of natural background bacteria was in all treatments 1.09±0.10 × 10⁶ cells ml⁻¹ (Mean±s.d.). The bacterial strains L. parvus, L. planktonicus, 2KL-27, 2KL-1, MoIso2 and Wo1 (for cell size and morphology see the inserted microphotograph, for further details see Table 1) were added to yield ∼25-fold natural background of bacterial biovolume present in the non-amended 5-μm filtrate used as control. Subsamples were collected in 12–24 h intervals.

Figure 2

Time-course changes in bacterial abundance in 5-μm treatments amended by bacterial strains of the genus Limnohabitans (a), that is, L. parvus, L. planktonicus, 2KL-27 and 2KL-1, and (b) with the strains MoIso2 and Wo1 compared to control with no bacteria added. Values are means for triplicates; error bars show s.d..

Figure 3

Time-course changes in HNF abundance and biovolume compared with bacterial biovolume in the treatments amended with respective bacterial strains (a–f, for further details see text to Figure 1 and Table 1) compared with control (g) with no bacteria added. Values are means for triplicates; error bars show s.d.. Values of growth efficiency, as % of bacterial biovolume introduced into the treatment at t₀, are listed in parenthesis for the treatments where positive HNF growth was detected (for details see Methods).

Figure 4

Comparison of HNF maximum growth rates (A), length of their lag phase after the treatment amendment (B) and the rate of HNF abundance increase growing on different bacterial strains (for further details see text to Figure 1 and Table 1) as related to the treatment with L. planktonicus (C) where the HNF abundance and biovolume peaked already at t₃₇ hours (compare Figure 3). Values are means for triplicates; error bars show s.d. Different letters indicate a significant difference (P<0.05, ANOVA, followed by Tukey's multiple comparison tests) between treatments amended with different bacterial strains.

Figure 5

A pie chart of the relative proportion (as %) of different subgroups of Stramenopiles within total Stramenopiles (accounting for 58–82% of the potentially bacterivorous flagellate groups) in treatments amended with different bacterial strains at times t₃₉ and t₆₃ compared with control treatments at t₀ and t₃₉ hours. For further details regarding the bacterial strains see text to Figure 1 and Table 1.

Figure 6

Absolute numbers of all Spumella-like flagellates (a) and of their three most abundant subclusters—Pedospumella, Spumella-like cluster D and Cluster E2 (b–d, respectively) in treatments amended with different bacterial strains at times t₃₇ and t₆₃ compared with control treatment. These data are based on the relative proportion of these subclusters within the target protistan groups (Figure 5 and Supplementary Figure 3) related to total HNF numbers (Figure 3). Note different y axis scaling for c, d. Values are means for triplicates; error bars show s.d.. For further details regarding the bacterial strains see text to Figure 1 and Table 1.