Prey-specific growth responses of freshwater flagellate communities induced by morphologically ...

35 Pages·2015·1.24 MB·English

AEM Accepted Manuscript Posted Online 15 May 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.00396-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Prey-specific growth responses of freshwater flagellate communities induced by
2 morphologically distinct bacteria from the genus Limnohabitans
3 Vesna Grujčić1,2, Vojtěch Kasalický1 and Karel Šimek1,2#
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5 1 Biology Centre CAS, v.v.i., Institute of Hydrobiology, Na Sádkách 7, CZ-37005 České
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6 Budějovice, Czech Republic o
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7 2 Faculty of Science, University of South Bohemia, CZ-37005 České Budějovice, Czech nlo
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8 Republic e
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10 Running title: Bacterial prey quality shape flagellate growth
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12 Keywords: Freshwater reservoir, flagellate bacterivory, Limnohabitans strains, bacterial food .
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13 quality, growth responses of flagellates m
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15 # Corresponding author: Karel Šimek n

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16 Biology Centre CAS, v.v.i., Institute of Hydrobiology r
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17 Na Sádkách 7, CZ-37005 České Budějovice, Czech Republic ,
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18 E-mail: [email protected]
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19 Phone: [phone] y

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25 ABSTRACT
26 Because their large growth potential is counterbalanced with grazing by heterotrophic
27 nanoflagellates (HNF), bacteria of the genus Limnohabitans, common in many freshwater
28 habitats, represent a valuable model for examining bacterial carbon flow to the grazer food
29 chain. We conducted experiments with natural HNF communities taken from two distinct
30 habitats, the meso-eutrophic Římov Reservoir and the oligo-mesotrophic Lake Cep (South D
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31 Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal n
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32 bloom and a late May clear water phase were each fed 3 Limnohabitans strains of differing d
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33 cell sizes. Water samples were pre-filtered (5 µm) to release natural HNF communities from f
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34 zooplankton control and then amended with the Limnohabitans strains: L. planktonicus, strain

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35 II-D5 (medium-sized, rod-shaped), strain T6-5 (thin long curved rod) and strain 2KL-3 (large p
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36 solenoid). Using temporal sampling and prey treatment we determined HNF growth e
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37 parameters such as doubling time, growth efficiency and length of lag phase prior starting a
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38 exponential growth. All three Limnohabitans strains supported HNF growth, however, in .
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39 significant prey-, site- and season-dependent fashions. For instance, addition of the /
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40 moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In A
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41 contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat il
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42 grazing-protected against smaller HNF, resulting in slower HNF growth and longer lag 2
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43 phases. These trends were particularly pronounced during the late May clear water phase,
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44 which was dominated by smaller HNF cells. This may indicate a longer “adaptation time” for g
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45 the flagellate communities toward the large prey size offered. s
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47 INTRODUCTION
48 Importance of both bacteria and their protistan grazers in planktonic microbial food webs has
49 been widely recognized (1, 2). Bacterioplankton transfers dissolved organic matter
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50 (DOM) to bacterial biomass that is consumed mainly by heterotrophic nanoflagellates (HNF)
51 or ciliates and channeled via grazing into the metazoan grazer food chain (3-5). The amount,
52 composition and temporal dynamics of DOM differs widely among the diverse set of
53 freshwater ecosystems and this DOM distinctly modulates the growth and population
54 dynamics of different bacterioplankton taxa (6). For instance, some bacterioplankton groups
55 such as Betaproteobacteria and Flavobacteria can respond to sudden pulses in algal-derived D
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56 organic carbon very rapidly, using doubling times that vary from several hours to days (7-10). n
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57 Representatives from a few highly abundant phylogenetic clusters of Betaproteobacteria d
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58 and Actinobacteria frequently dominate bacterioplankton populations in a wide variety of f
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59 freshwater ecosystems (11-13). Among the Betaproteobacteria, Limnohabitans and

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60 Polynucleobacter have been documented as the most ecologically important genera (6, 14- p
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61 18). The Limnohabitans genus is composed of five major lineages. Four are the so-called R- e
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62 BT bacteria, that are identified via hybridization with the R-BT065 phylogenetic probe (19), a
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63 and one lineage, known as LimA, that does not hybridize with the probe (18). The four .o
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64 lineages of the R-BT cluster are represented by rapidly growing, morphologically diverse /
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65 phylotypes with generally opportunistic life strategies (18, 20, 21). They also display a high A
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66 metabolic flexibility in incorporating simple organic substrates (18). Limnohabitans bacteria il
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67 from the R-BT cluster are omnipresent in a wide variety of pH-neutral or alkaline freshwater 2
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68 habitats (16). However, they have been also found as microflora in the digestive tract of
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69 algivorous Daphnia magna (22), or as epibionts in filter combs of Daphia galeata (23). Thus g
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70 it seems that the main substrates supporting their rapid growth in situ are algal-derived s
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71 substances (19, 24, 25). This has also been suggested for several phylogenetically narrow
72 lineages of Flavobacteria (6, 7, 9). Representatives of both the Flavobacteria and the
73 Limnohabitans genera are dominated by larger cell sizes (9), yet are within the edible size
74 range for HNF (26, 27). Notably, it has been shown that the high growth rates of
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75 Limnohabitans bacteria are counterbalanced by considerable loss rates via grazing because
76 these bacteria are selectively grazed by flagellates (9, 28). It is because of these specific
77 ecophysiological traits, high growth rate and high grazing pressure, that Limnohabitans
78 bacteria likely have a prominent role in carbon transfer to higher trophic levels.
79 In several lakes, remarkable changes in relative contributions of the total R-BT bacteria
80 or their specific genotypes to the total bacterioplankton community were found to occur D
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81 during the spring phytoplankton bloom and the clear water phase (6, 9). In addition to n
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82 considerable shifts in bacterioplankton community composition, there were also changes in d
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83 HNF numbers and their size class distribution. During spring bloom periods modest top-down f
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84 control of the HNF population usually allows for their development of abundant populations.

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85 Generally, large cell sizes of bacteria also prevail with marked contributions from both p
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86 Limnohabitans and Betaproteobacteria (9). This sharply contrasts to the clear water phase e
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87 when plankton trophic structure is strongly top-down controlled by larger filter-feeding a
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88 zooplankton (29). Here, HNF populations are heavily decimated and dominated by small cells .
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89 and the absolute and relative contributions of the large cell Betaproteobacteria decreases /
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90 along with a general shift toward smaller cells within the entire bacterioplankton (4, 9). A
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91 Notably, natural communities of bacterivorous HNF have large growth potential (20, 30) il
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92 and flagellate predator–bacterial prey relationships are so flexible that sudden changes in the 2
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93 prey community may induce very rapid changes in the flagellate predator community (31).
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94 Feedback control of bacterial prey food quality on predator community composition and g
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95 growth are potentially important but are largely unstudied. This is mainly due to methodical s
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96 problems and a lack of representative isolates from relevant bacterioplankton genera. Previous
97 experiments (18) have documented rapid growth of HNF on four medium-sized but
98 morphologically different Limnohabitans strains. Strains with distinct morphologies yielded
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99 differences in HNF growth and community parameters, which could not be related just to size
100 differences among the bacterial prey (18).
101 In this study, we report results from experiments in which three strains of
102 Limnohabitans of medium to large cell volumes were introduced into natural HNF
103 communities collected in parallel from a meso-eutrophic freshwater reservoir and an oligo-
104 mesotrophic sand-mining pit lake in two distinct seasonal phases (spring bloom versus clear D
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105 water phase). We hypothesized that different environmental scenarios, sudden pulses in n
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106 availability of bacterial prey of high food quality may yield different effects on the growth d
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107 parameters of bacterivorous HNF and thus also alter the rate of carbon flow into the grazer f
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108 food chain. In four experiments we exploited predator-prey manipulations to address the

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109 following aims: Examination of the community growth parameters, such as doubling time, lag p
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110 phase duration and flagellate gross growth efficiency, within freshwater HNF assemblages e
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111 feeding on Limnohabitans strains of different cell size and morphology, and examination of a
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112 how the different trophic status and contrasting seasonal phases of the two study sites might .
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113 modulate growth responses of their indigenous HNF populations. /
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114 MATERIALS AND METHODS p
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115 Experimental organisms and sampling sites. Three Limnohabitans strains (L. planktonicus
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116 II-D5 (32) and two undescribed Limnohabitans sp. strains - 2KL-3 and T6-5 (18)), of 9

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117 markedly different size and shape, were used in four prey-enrichment experiments with the
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118 same experimental design (for details see Table 1 and Fig. 1). e
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119 Water samples with natural HNF communities were collected from two distinct
120 freshwater habitats in South Bohemia (Czech Republic): Římov reservoir (48°50’46.90"N,
121 14°29’15.50"E), a meso-eutrophic drinking water reservoir of circum neutral pH (for more
122 details see (23) and references therein) and Lake Cep, a sandpit lake (48°92’49.24"N,
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123 14°88’68.11"E) created as a consequence of past sand-mining in this area. The lake is
124 generally poor in nutrients (Table 2) and considered oligo-mesotrophic.
125 Design of experiments. Two experiments were conducted during two different phases of
126 plankton succession in both study sites in parallel. The first experiment was run from 23 till
127 28 April 2012 (water temperature - Římov 16.6ºC, Cep 17ºC), during the spring
128 phytoplankton bloom period. The second experiment was run from 28 May till 2 June 2012 D
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129 (Římov 19ºC, Cep 21ºC) and coincided with the late clear water phase of plankton succession n
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130 in both habitats. The temperature of the water at both study sites was measured on the day of d
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131 water sampling. All treatments were incubated at 18 ºC which closely resembled the in situ f
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132 temperatures.

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133 Plankton samples (10 L) were taken from each habitat (0.5 m depth) 24 hours before the p
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134 experiments started. Water (6 L) from each habitat was gravity filtered through 5-µm pore- e
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135 size filters (147-mm diameter) to release the HNF from zooplankton grazing pressure. Note a
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136 that most bacterivorous HNF cells are smaller than 5 µm and thus the filtrate contained a .
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137 representative site-specific community of HNF. The 5-µm filtrates containing the HNF /
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138 communities were incubated at 18º C for 24 hours. The incubation yielded approximately A
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139 two-fold increases in HNF abundance, and slight decreases in the the number of free-living il
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140 bacteria with most remaining bacteria (~ 1 x 106 ml-1) in the form of flocks or filaments 2
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141 inedible for these HNF populations.
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142 All three Limnohabitans strains were pre-grown in nutrient-rich liquid medium (3 g L-1 g
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143 NSY) (33). Twenty four hours before starting the experiment these Limnohabitans strains s
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144 were concentrated by centrifugation (20 min, 5000 x g) and the pellets were resuspended in
145 0.2-µm filter and then sterilized water from both habitats (31). This procedure did not appear
146 to affect bacteria viability as some of the strains grew after inoculation into experimental
147 treatments with natural water from either habitat (Figs. 2, 3).
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148 As necessary, sterile, bacteria-free water from each habitat (0.2-µm filtrated) was used to
149 dilute the 5-µm fractions with growing HNF communities to obtain solutions of the same
150 initial concentration of HNF (2 x 103 ml-1). The 5-µm filtrates with natural HNF communities
151 (300 ml aliquots) were placed in sterile 500 ml Erlenmeyer flasks. HNF populations were
152 then amended with solutions of prey bacteria. Because the prey bacteria differed markedly in
153 cell volume (Table 1), the additions of the morphologically distinct Limnohabitans strains D
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154 were set to yield approximately the same initial volume of biomass for all three strains (31). n
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155 Experiments were run in triplicate and treatments were kept at 18º C in dark. The treatments d
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156 containing only natural bacteria and HNF present in the original samples from both sites f
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157 served as controls compared to the prey enriched treatments (referred to as II-D5, 2KL-3 and

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158 T6-5 throughout the text). Subsamples for detecting HNF and bacterial abundance and p
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159 biomass were aseptically taken within a laminar flow hood in 12-24 hours intervals. At e
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160 selected time points (t t t and t ) additional samples for fluorescence in situ a
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161 hybridization (FISH) were collected. The detailed outline of the experimental set-up is .
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162 depicted in Figure 1. /
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163 Bacterial and heterotrophic flagellates enumeration and biovolumes. Samples fixed A
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164 with formaldehyde (2% final concentration) were used for enumeration of bacteria (0.5-2 ml il
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165 subsamples) and HNF (4-10 ml subsamples) on 0.2-µm and 1-µm pore-sized filters 2
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166 (Osmonics, MN, USA), respectively. All samples were stained with DAPI (4’, 6-diamidino-
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167 2-phenylindole) and the microbes were counted via fluorescence microscopy (34). Bacterial g
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168 biovolume was measured by using semiautomatic image analysis system (NIS-Elements 3.0, s
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169 Laboratory Imaging, Prague, Czech Republic). To calculate mean cell volumes of HNF
170 (approximated to prolate spheroids, see also (31)), lengths and widths of 50 cells in each
171 triplicate treatment were measured manually on-screen with a built-in tool of a PC-based
172 image analysis system (NIS-Elements 3.0).
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173 Net HNF growth rate was calculated based upon the equation for the exponential growth:
174 μ = (lnN – lnN )/(t – t ) , where μ is the specific growth rate (day-1), N and N are HNF
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175 abundances (cells ml-1) at time t and time zero t (day), respectively, ln is natural logarithm.
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176 The three consecutive time points on the HNF growth curve yielding the largest r2 were
177 selected for calculation. The parameter µ allowed for calculating HNF doubling time (20)
178 used as a growth metric throughout the text. D
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179 The lag phase was calculated as the period from the time zero to the intercept between the n
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180 best fit line of HNF growth and the zero-time level of HNF abundance. d
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181 Growth efficiency calculation of HNF is based on cellular biovolume as follows: f
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182 Growth efficiency = (net HNF biovolume increment/bacterial biovolume introduced) x 100,

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183 where the net HNF increment is the maximum HNF biovolume achieved minus the HNF p
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184 biovolume present at t0; and the net HNF biovolume increment is divided by bacterial e
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185 biovolume introduced into a treatments at t . a
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186 Additionally, relative growth rates were calculated. Rates of a HNF abundance increase .
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187 growing on different bacterial strains were related to the treatments amended with the /
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188 bacterial strain T6-5, where HNF populations generally displayed the most rapid growth. A
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189 Thus, the HNF abundance in T6-5 treatments (peaking at t66 h) was set as 100% for il 9
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190 comparison of the abundance increases in other treatments. 2
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191 Chemical analyses – carbon, phosphorus and chlorophyll a concentrations. Water
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192 used for the experiments was analyzed at the time of the sample collection at the study site g
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193 (Table 2). Dissolved organic carbon (DOC) was analyzed in samples filtered through glass- s
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194 fiber filters of 0.4 μm nominal pore size (GF-5, Macherey-Nagel) with a TOC 5000A analyzer
195 (Shimadzu). Dissolved reactive phosphorus (DRP as PO -P) was analyzed according to
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196 Murphy & Riley (35). Total phosphorus (TP) was determined by the molybdate method
197 following perchloric acid digestion according to Kopáček and Hejzlar (36). Chlorophyll a
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198 (Chl-a) concentrations were determined spectrophotometrically after the extraction with
199 acetone (37).
200 Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). The
201 CARD-FISH protocol for in situ hybridization (38) was used with the BET42a and R-BT065
202 oligonucleotide probes targeting all Betaproteobacteria (BET, (39)) and its R-BT subcluster
203 (R-BT (34)), respectively. The probe R-BT065 covers all three strains from the D
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204 Limnohabitans lineages used in these experiments (18). We examined proportions, in n
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205 duplicate, of the probe-targeted bacteria in plankton of the study sites at the time of sample d
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206 collection (t0), and in all experimental treatments at times t24, t48 and t66 h. Moreover, for fr
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207 verifying the presence of the prey bacteria in HNF food vacuoles, the CARD-FISH protocol

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208 detailed by Jezbera and coworkers (40) was applied to detect the ingested bacterial prey. p
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209 Statistical analysis. Statistical analyses were performed with JMP v.8. (2008, SAS e
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210 Institute, Cary, NC, USA). Using three-way ANOVA, we analyzed the effects of two distinct a
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211 phases of plankton succession, study site and strains characteristics on HNF growth .
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212 efficiency, doubling time (DT), lag-phases, and % of abundance increase of T6-5 at t66 hours. o/
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213 When these ANOVAs were significant, Post hoc tests were used to determine differences A
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214 among means (Student test when we compared only two means and Tukey test when we il
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215 compared three or more mean values). Canonical correspondence analysis (CCA) was 2
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216 performed with Excel stats package. We tested if growth parameters (doubling time, lag-
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217 phase, and growth efficiency) varied with study site, month, and bacterial strain. g
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218 Results s
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219 Water chemistry and Chl-a concentrations. Generally, phosphorus and Chl-a
220 concentrations indicated that Římov was a more productive environment than oligo-
221 mesotrophic Lake Cep (for details see Table 2). However, during the May experiments (late
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222 clear water phase) all chemical parameters and Chl-a dropped dramatically in Římov, while
223 no obvious trends were observed in Lake Cep.
224 Temporal change in bacterial numbers as a response to HNF grazing. Prey
225 availability was suddenly increased by about one order of magnitude and thus growth
226 responses of HNF communities were clearly attributable to the particular, introduced prey
227 (Figs. 2-6). Generally, in most prey-amended treatments bacterial numbers started to decrease D
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228 markedly after approximately 40-66 hours (Figure 2), whereas in II-D5 treatments in Lake n
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229 Cep samples a more rapid abundance drop occurred within the first 40 h. In contrast, bacterial d
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230 numbers stayed relatively stable in most T6-5 and 2KL-3 treatments between t0-53 h. However, fr
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231 unique trends were observed in the treatments T6-5 (April-Římov) and 2KL-3 (May-Římov),

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232 with remarkable increases in bacterial numbers occurring mainly between t h, which was p
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233 before the onset of HNF exponential growth (Figs. 2 and 3). These data indicated growth of e
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234 the treatment-added bacteria during the initial stage of the experiment. After 50-90 hours, the a
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235 growing HNF populations had decimated bacteria abundances in all treatments to levels .
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236 comparable to the control treatments. Notably, the relative proportions of RBT bacteria, which /
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237 likely indicated a decay rate in the added prey, dropped dramatically from the initial ~94-98% A
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238 at t0 to treatment-specific averages ranging between 5 to12% of total bacteria by t66 h (data not il 9
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239 shown). All introduced prey items were clearly detected in flagellate food vacuoles based 2
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240 upon their size, morphology and hybridization with the R-BT065 FISH-probe as shown in
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241 Figure 4. In contrast to prey-amended treatments, no obvious trends in bacterial abundance g
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242 and biomass were observed in control treatments (Figs. 2 and 3). s
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243 Growth of flagellates on different Limnohabitans strains. Remarkable growth of HNF
244 populations occurred in the Limnohabitans prey-amended treatments, with profound prey-,
245 site- and distinct seasonal phase-specific differences (Fig. 3). HNF abundances increased from
246 2 x 103 cells ml-1 to treatment-specific maxima ranging from 30 to even 90 x 103 cells ml-1
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