Toxicity of violacein-producing bacteria fed to bacterivorous freshwater plankton

Transcription

1 Limnol. Oceanogr., 54(4), 2009, E 2009, by the American Society of Limnology and Oceanography, Inc. Toxicity of violacein-producing bacteria fed to bacterivorous freshwater plankton Peter Deines, 1 Carsten Matz, 2, * and Klaus Jürgens 3 Department of Physiological Ecology, Max Planck Institute for Limnology, Plön, Germany Abstract Chemical defenses have been hypothesized to be widespread among aquatic bacteria, but few studies have assessed the susceptibility of pelagic consumers to bacterial secondary metabolites. In this study, we determined the effect of bacteria containing the indole alkaloid violacein on a range of freshwater plankton organisms that ingest bacteria. Growth and survival of two nanoflagellates (Bodo saltans, Ochromonas sp.), two ciliates (Colpidium campylum, Tetrahymena pyriformis), two rotifers (Keratella cochlearis, Brachionus calcyfloris), and adults and juveniles of the cladoceran Daphnia magna were examined in laboratory experiments in which these organisms were fed bacterial diets supplemented with violacein-producing bacteria such as Janthinobacterium lividum and Chromobacterium violaceum. Unselective uptake of violacein-producing bacteria resulted in significantly lower survivorships, indicating acute toxicity to all zooplankton taxa tested. The presence of 10% C. violaceum in an otherwise nontoxic bacterial diet, equivalent to 9 ng violacein ml 21, reduced growth rates and peak abundances of flagellates, ciliates, and rotifers by %. For D. magna, wild-type C. violaceum was poisonous starting at concentrations, cells ml 21, whereas a violacein-deficient mutant did not have any lethal effect on the animals. Species-specific variability in predator susceptibility was explained by the negative correlation between toxin sensitivity and biomass-specific clearance rates. The susceptibility of a broad spectrum of planktonic bacterial consumers to violacein-producing bacteria illustrates the potential effect of bacterial bioactives on the structure of aquatic food webs, biodiversity, and ecosystem functioning. Introduction Secondary metabolites and chemical defenses are major determinants of trophic interactions in marine benthic communities (Hay and Fenical 1988). Following the general hypothesis that predator-active metabolites should be evolutionarily favored in top-down controlled populations, it has been increasingly recognized that chemical antagonisms are widespread in the planktonic food webs of marine and freshwater ecosystems (Verity and Smetacek 1996; Turner and Tester 1997; Wolfe 2000). Several classes of phytoplankton are known to include taxa that are inhibitory or toxic to herbivores (Cembella 2003). Aldehydes extracted from bloom-forming diatoms have been shown to impair the reproductive success of copepods (Ianora et al. 2004; Pohnert 2005), thereby exemplifying the progress being made in understanding the chemical mechanisms that structure pelagic ecosystems. The elucidation of chemically mediated algae zooplankton interactions is anticipated to increase our basic understanding of the structural dynamics in pelagic food webs and ecosystem function. In addition to the consumption of microalgae, bacterivory is an integral part of the pelagic food web and thus a * Corresponding author: carsten.matz@helmholtz-hzi.de Present address: 1 Biological & Environmental Systems Group, Department of Chemical and Process Engineering, The University of Sheffield, Sheffield, UK 2 Department of Cell Biology, Helmholtz Center for Infection Research, Braunschweig, Germany 3 Leibniz Institute for Baltic Sea Research, Rostock-Warnemünde, Germany 1343 driving force behind the biogeochemical cycling of organic matter in the water column (Strom 2000). The major planktonic bacterivores are heterotrophic and mixotrophic nano- and microplankton (Sherr and Sherr 2002). However, many taxa of herbivorous filter-feeding metazooplankton are in fact omnivorous and as such also have bacteria within their diets (Jürgens 1994; Pace and Cole 1996). The fact that bacterial prey is utilized by bacterivores at different trophic levels suggests that the qualitative composition of bacterioplankton directly affects the structure of planktonic food webs. Consistent with their remarkable biosynthetic versatility, aquatic bacteria produce an enormous range of secondary metabolites, of which significant fractions are biologically active toward pro- and eukaryotes (Jensen and Fenical 1994). The ever-increasing list of bioactive bacteria isolated from diverse aquatic habitats indicates that chemical antagonisms, including defense against predators, play a significant role in aquatic ecosystems (Long and Azam 2001; Matz and Kjelleberg 2005; Ianora et al. 2006). Moreover, bacteria have been discussed as the producers of bioactive metabolites isolated from algal blooms (Doucette 1995). In contrast to recent interest in the chemical defenses of phytoplankton, the defensive function of bacterial metabolites and their effect on bacterivores in aquatic food webs have barely been explored. Grazing enrichments of bacterioplankton from a mesotrophic lake have recently revealed the presence of bacteria of the genus Janthinobacterium (Matz 2002). Studies of the isolate Janthinobacterium lividum strain CM37 identified violacein as a potent toxin against bacterivorous nanoflagellates (Matz et al. 2004). Violacein is a purple-colored pigment characterized as an L-tryptophan derived alkaloid consisting of three structural units: 5-hydroxyindole, 2-

2 1344 Deines et al. such as lake bacterioplankton (Matz et al. 2004), rivers (Halda-Alija and Johnston 1999), and activated sludge (Curds and Vandyke 1966). The acute toxic effect of violacein-producing bacteria (VPB) on heterotrophic nanoflagellates (HNFs; Matz et al. 2004) and the putatively widespread occurrence of predator-active molecules in aquatic bacteria (Matz et al. 2008) raise the question as to whether the presence of bioactive bacteria affects the fitness and mortality of bacterial consumers other than HNFs. In an initial effort to determine the potential effect of bioactive bacteria on planktonic food webs, we conducted laboratory experiments in which the major groups of bacterivorous freshwater zooplankton were exposed to a VPB-containing diet. Specifically, strains of VPB and non-vpb were used in experiments examining the growth and survival of the zooplankton as well as their ingestion of mixtures of toxic and nontoxic bacteria. Methods Zooplankton stock cultures In our assays, representatives of the four major groups of bacterivores commonly found in lake zooplankton were used (i.e., nanoflagellates, ciliates, rotifers, and cladocerans). All stock cultures were kept at 20uC. Fig. 1. Production of the toxic bacterial metabolite violacein. (a) Cellular violacein content of J. lividum CM37 (JL+), C. violaceum CV017 (CV+), and C. violaceum CV026 (CV2) and the chemical structure of violacein. (b) Negative relationship between the violacein content of the bacterial strains offered as food to the nanoflagellate Ochromonas sp. and the resulting growth rate of the flagellate (growth rate violacein content ). Violacein content of the bacterial diet was calculated from bacterial cell numbers and the violacein concentration of quantitative cell extractions. Shown are means 6 SD. pyrrolidone, and oxindole (Fig. 1a). Although it elicits antibiotic activities against a range of microorganisms (Nakamura et al. 2002), violacein has recently been shown to act specifically as an antipredator metabolite in marine bacteria (Matz et al. 2008). Bacteria containing nanomolar concentrations of violacein inhibit flagellate feeding and induce a conserved eukaryotic cell death program (Matz et al. 2004, 2008). Intriguingly, violacein is produced by at least four bacterial genera: Chromobacterium, Janthinobacterium, Pseudoalteromonas, and Microbulbifer (Sneath 1956; Matz et al. 2008). Whereas Pseudoalteromonas spp. and Microbulbifer species are widely distributed in marine ecosystems (Skovhus et al. 2004; Matz et al. 2008), bacteria of the genera Janthinobacterium and Chromobacterium have been repeatedly isolated from freshwater habitats, Flagellates The heterotrophic flagellate Bodo saltans (Kinetoplastida) and the mixotrophic flagellate Ochromonas sp. (Chrysomonadida) had been isolated from mesotrophic lakes in Germany (Matz and Jürgens 2001). Multiclonal cultures were kept in the dark and treated with bacterial antibiotics to reduce the number of background bacteria. Monoxenic stock cultures were maintained in erlenmeyer flasks in modified Woods Hole (WC) medium (equivalent to Chu-12 medium supplemented with thiamin and biotin) and amended with Pseudomonas putida MM1 (Matz and Jürgens 2001) and glucose at a concentration of 100 mg L 21. For the experiments, flagellates were taken from 5-d-old stock cultures when their abundances reached about 10 6 cells ml 21 and the number of bacteria was reduced below 10 4 cells ml 21. Ciliates Multiclonal cultures of the ciliates Colpidium campylum and Tetrahymena pyriformis had been obtained from the culture collection of the IGB-Berlin (Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany; courtesy of V. C. L. Meyer). According to the instructions of the culture collection, both stock cultures were maintained on bottled spring water (we used VolvicH water; contains: Ca 2+,Mg 2+,Na +,K +,Cl 2, SiO 2, SO 2{ 4,HCO { 3 ) amended with P. putida MM1 growing on yeast extract and dried banana skin. Ciliate cultures were kept in cell culture flasks (Sarstedt) in the dark and were transferred into fresh medium once a week. Before the experiments, ciliate stock cultures were washed four times by centrifugation (10 min at 1500 revolutions per minute [rpm]) to separate ciliates from bacteria and to give a final ciliate abundance of cells ml 21.

3 Bioactive bacteria affect zooplankton 1345 Rotifers The rotifers Keratella cochlearis and Brachionus calyciflorus (both from the culture collection of the IGB-Berlin) were kept in cell culture flasks (Sarstedt) in bottled spring water (VolvicH) supplemented with the small cryptophyte Cryptomonas sp. (concentration, 1 mg C L 21 ; culture collection of the Max Planck Institute for Limnology [MPIL]). Rotifer cultures were kept in dim light (10 30 mmol photons m 22 s 21 ) under a 12 : 12 h light : dark (LD) cycle and transferred into fresh medium every 3 d. For the experiments, rotifers were picked individually and transferred to tissue cell culture plates containing bottled spring water. Daphnids Clonal cultures of the cladoceran Daphnia magna (culture collection of the MPIL; originally isolated from Großer Binnensee [Germany]) were kept in lake water that had been filtered through a 0.45-mm membrane filter. The chlorococcal algae Scenedesmus obliquus Meyen (culture collection of the MPIL), grown in a chemostat on Chu-12 medium (equivalent to WC medium but lacking vitamins thiamin and biotin), served as the food source. D. magna stock cultures were kept in 1.5-liter glass beakers and maintained by adjusting the algal concentration daily to 1 mg C L 21. As rotifers, Daphnia Scenedesmus cocultures were kept in dim light (10 30 mmol photons m 22 s 21 ) under 12 : 12 h LD. Bacterial stock cultures J. lividum CM37 was isolated from low-nutrient laboratory mesocosms containing 3-mm filtered plankton samples from a mesotrophic lake (Schöhsee, northern Germany; Matz 2002). Chromobacterium violaceum (CV) had originally been isolated from freshwater (Sneath 1956). Strain CV017 produces copious amounts of the purple pigment violacein, whereas CV026 is nonpigmented because of a mutation in the regulator gene cvii of the cvii/cvir quorum-sensing system (Blosser and Gray 2000). The well-characterized food bacterium P. putida MM1 (Matz et al. 2004) served as a non-toxic reference strain of high food quality. Bacteria were routinely grown on yeast extract (5 g L 21 )at20uc. Before the feeding experiments, bacteria were harvested by centrifugation (12,000 rpm for 15 min), washed, and resuspended in the culture medium of the respective bacterivore to be tested. To determine the amount of violacein produced by bacterial strains, we followed the method described by Blosser and Gray (2000). In short, violacein was extracted from resuspended cell pellets by a combined treatment of sodium dodecyl sulfate (final concentration 5%) and watersaturated butanol. Violacein was quantified photometrically by measuring the absorbance at a wavelength of 585 nm. Cellular violacein contents were calculated from a previously established calibration curve between absorbance and purified violacein. Experiments on zooplankton survivorship All survival experiments were performed with the same medium and culture conditions as described above for maintenance of stock cultures. Batch culture experiments were carried out to examine the population response of flagellate, ciliate, and rotifer species to different concentrations of the violacein-producing strain C. violaceum CV017. This was tested in four mixtures of the nontoxic P. putida MM1 containing an increasing proportion of C. violaceum CV017 (0%, 10%, 25%, and 50%). The total concentration of the added bacteria was 10 7 cells ml 21 in the flagellate and rotifer experiments and 10 8 cells ml 21 in the ciliate experiments. Flagellate and ciliate experiments were carried out in erlenmeyer flasks, and cell numbers were determined from three replicates for each treatment, lasting for at least 50 h. Rotifer experiments were performed in tissue cell culture plates (well diameter 22.2 mm, volume 6.30 ml; Techno Plastic Products) containing 6 10 animals per well. Because of the low number of individuals per well, each treatment of the rotifer assays was run in replicates of 24. To exclude maternal effects on toxin susceptibility, D. magna juveniles and adults were derived from neonates of the fourth generation of offspring. The susceptibilities of adults and juveniles to pure bacterial suspensions was compared in acute toxicity tests at eight different concentrations of strain CV017 (0, , , , , and cells ml 21 ) over 24 h. Long-term effects of a mixed diet containing food algae and strain CV017 were tested by growing juvenile D. magna on 1 mg C L 21 S. obliquus supplemented with two concentrations of the toxin-producing strain ( cells ml 21 and cells ml 21 ). Control treatments included a nonfood control (filtered lake water) and 1 mg C L 21 S. obliquus supplemented with the nontoxic strain CV026 ( cells ml 21 ). D. magna individuals were kept in separate beakers (200 ml), inspected every 6 12 h, and transferred daily into freshly prepared suspensions over a period of 10 d. Treatments in each of the experiments were replicated with fifteen individuals. Ingestion experiments Feeding rates of Ochromonas sp., T. pyriformis, andb. calcyflorus were determined on a 1 : 1 mixture of strain CV017 with the nontoxic reference strain P. putida MM1. The two bacterial strains were added to a final concentration of cells ml 21. The experiment was terminated for the flagellate after 10 min and for the ciliate and the rotifer after 22 h (in each case by adding icecold glutaraldehyde, 2% final concentration). Bacteria were quantified by immunofluorescence microscopy with the use of polyclonal antibodies against C. violaceum CV017 and P. putida MM1 (Matz et al. 2004). Bacteria ingested by Ochromonas sp. were directly detected in the flagellate food vacuole (Matz et al. 2004). For T. pyriformis and B. calcyflorus, uptake rates were calculated from the relative numbers of suspended bacteria. Ingestion experiments were run in triplicate and performed with the same medium and culture conditions as described above for maintenance of stock cultures. Enumeration Cell numbers of bacteria, flagellates, and ciliates were determined from formaldehyde-fixed (2%) samples. These were stained with 49,6-diamidino-2-phenylindole (DAPI), and the cells were counted by epifluorescence microscopy. For the enumeration of surviving flagellate and ciliate cells, only those with intact cell

4 1346 Deines et al. boundaries were counted because dead cells showed diffuse structures as the result of lysis (Matz et al. 2004). Rotifers and daphnids were counted live during the experiment at different time intervals. Individuals were considered dead after showing no swimming activity despite repeated disturbance. Data analysis Exponential growth curves of flagellates and ciliates were used to calculate the population growth rate according to m 5 (ln N 1 2 ln N 0 )/t, where N 0 and N 1 were the cell concentrations at the beginning and after time interval t. In the feeding experiments, the ingestion rate I (bacteria individual 21 h 21 ) was measured, and the clearance rate F (ml individual 21 h 21 ) was calculated by the following formula: F 5 I/B (nl individual 21 h 21 ), where B refers to the bacterial concentration (cells ml 21 ) used. Food selection was quantified by the selectivity coefficient a (Chesson 1983). The selectivity index a cv for predation on C. violaceum was calculated according to the equation a cv 5 a cv /(a cv + a mm ), where a cv and a mm are the clearance rates for C. violaceum and P. putida MM1. The index ranged from 0 (uptake of only P. putida MM1) to 1 (exclusive uptake of C. violaceum), with a value of 0.5 indicating nonselective feeding. LT 50 values (median lethal time) were determined in the survival experiments to describe the time needed to kill 50% of the initial population. LC 50 values (median lethal concentration at which the number of animals is decreased by 50%) were estimated for D. magna juveniles and adults in the acute toxicity test. One-way ANOVA was used to compare differences between bacterial strains, growth, and feeding rates. Differences in the species numbers over time were tested with repeated measures ANOVA followed by a post hoc test of the means after Tukey. The significance of possible differences between survival functions was determined by the log-rank test. To quantify the effect of CV017 or starvation on Daphnia survival, the time needed to kill 50% of the animals (LT 50 ) was calculated as the median of the Kaplan Meier survival function estimation. Linear regression models were used to analyze the relationship between violacein content and flagellate growth rates and between LT 50 values and biomass-specific clearance rates. The data were analyzed with STATISTICA (version 5.1; StatSoft). Results Violacein production by freshwater bacteria Lake bacterioplankton growing in laboratory mesocosms over a 2- month period were dominated by the two bacterivorous nanoflagellates Ochromonas sp. and Spumella sp. Video microcopy based analysis of grazing-protected bacterial phenotypes revealed that a proportion of bacteria caused nanoflagellate cells to lyse on ingestion. These observations co-occurred with the isolation of the violacein-producing bacterium J. lividum from the mesocosms. The cellular content of J. lividum strain CM37 was up to fg cell 21, which was about 2.5-fold higher than for another freshwater isolate, C. violaceum CV017 (Fig. 1a). Differences in the violacein content of J. lividum CM37, C. violaceum CV017, and the violacein-negative strain C. violaceum CV026 were significant (Fig. 1a; F 2, , p, ). By testing the effect of these strains on the flagellate Ochromonas sp., we found a significant negative correlation between the violacein content of the bacterial strains fed and flagellate growth rates (Fig. 1b; R , F 1, , p, ). Because J. lividum repeatedly lost its ability to produce violacein after prolonged growth in laboratory culture, we chose to continue our studies with C. violaceum. The availability of the violacein-negative strain CV026 for C. violaceum, allowed us to conduct a series of defined feeding experiments on the effects of a bacterial secondary metabolite on bacterivorous and omnivorous zooplankton. Survivorship experiments Flagellate survival: We tested the susceptibility of two nanoflagellates to VPB by offering four different food ratios of VPB and nontoxic P. putida MM1 (0% C. violaceum, 0 ng violacein ml 21 ; 10% C. violaceum, 9.1 ng violacein ml 21 ; 25% C. violaceum, 22.7 ng violacein ml 21 ; and 50% C. violaceum, 45.4 ng violacein ml 21 ). Increasing proportions of the violaceinproducing C. violaceum strain CV017 in the offered bacterial diet negatively influenced the survival response of flagellates, as shown for B. saltans (Fig. 2a). In the 0% CV017 treatment (100% P. putida MM1), B. saltans populations showed an increase by about 70%. The presence of 10% of the violacein-producing CV017 resulted in significantly reduced growth rates of the nanoflagellates B. saltans and Ochromonas sp. (Table 1). Peak abundances of flagellates were significantly reduced by 19 29% in the 10% CV017 treatment compared with the 0% CV017 treatment. LT 50 values determined on a 25% CV017 diet showed that half of the flagellate populations were killed within h of incubation. Ciliate survival: As observed for the flagellates, growth rates of both ciliate species C. campylum and T. pyriformis were significantly lower on the 10% C. violaceum CV017 diet (9.1 ng violacein ml 21 ) than on the 0% CV017 diet (Table 1). Although numbers of C. campylum on a violacein-negative diet almost doubled within 30 h (Fig. 2b), high growth rates were completely compensated by the presence of 9.1 ng violacein ml 21, leading to negative growth. In the 10% CV017 treatment, maximum abundances of both ciliates were significantly reduced by 18 68% compared with the 0% CV017 treatment. Although bacterial diets containing CV017 were also lethal for T. pyriformis, toxin sensitivities as determined by LT 50 values differed considerably between the two ciliate species C. campylum and T. pyriformis ( h and h, respectively). Rotifer survival: The two rotifer species K. cochlearis and Brachionus calcyfloris were able to maintain their initial population size on a diet of 100% P. putida MM1 (0% CV017; Table 1). As observed for flagellates and ciliates, rotifer survivorship declined as the proportion of the violacein-producing strain CV017 increased in their diets (Fig. 2c). Populations of K. cochlearis feeding on 10%, 25%, and 50% CV017 were reduced to extinction within

5 Bioactive bacteria affect zooplankton 1347 showed that half of the population of rotifers was killed within h of incubation. Survival of adult and juvenile daphnids: The effect of VPB on adult and juvenile individuals of the cladoceran D. magna was tested in coculture with the green algae S. obliquus. The number of D. magna individuals remained stable over a period of 132 h throughout cultivation on S. obliquus, as well as on a mixed diet consisting of S. obliquus and the violacein-deficient strain CV026 (Fig. 3a). Likewise, D. magna offspring size was comparable in these two treatments, reflecting its ability to grow and reproduce on a nontoxic bacterial diet (data not shown). The presence of the violacein-producing strain C. violaceum CV017 at a concentration of cells ml 21 (which is equivalent to 18.1 ng violacein ml 21 ), however, led to a dramatic drop in D. magna survivorship after 48 h (LT h). The response to a concentration of CV017 cells ml 21 (90.7 ng violacein ml 21 ) was immediate and resulted in LT h. Acute toxicity tests of D. magna over a range of bacterial concentrations revealed different sensitivities for juvenile and adult individuals toward a diet containing 100% C. violaceum CV017 (Fig. 3b). Juvenile mortality increased strongly at bacterial concentrations of around ml 21, with 100% mortality reached at bacteria ml 21, whereas adult D. magna reached 100% mortality at a concentration of cells ml 21. The higher susceptibility of juvenile individuals was also illustrated by the lower LC 50 value of bacteria ml 21 (18.1 ng violacein ml 21 ) compared with bacteria ml 21 (145.1 ng violacein ml 21 ) for D. magna adults. Ingestion rates and feeding preferences The flagellate Ochromonas sp., the ciliate T. pyriformis, and the rotifer B. calcyfloris did not stop feeding when offered a mixed bacterial diet of 50% C. violaceum CV017 and 50% P. putida MM1. Feeding selectivity indices determined for Ochromonas sp. and T. pyriformis were not significantly different from 0.5 and therefore indicated that neither the flagellates nor the ciliates preferred nontoxic MM1 over violacein-producing CV017 (Table 1). A discrimination against C. violaceum CV017 was also not observed for the rotifer B. calcyfloris. Fig. 2. Growth and survival of (a) the flagellate B. saltans, (b) the ciliate C. campylum, and (c) the rotifer K. cochlearis at increasing proportions of the violacein-producing C. violaceum strain CV017 in the bacterial diet. Strain CV017 was mixed with nontoxic P. putida MM1 in four ratios (0%, 10%, 25%, and 50% of CV017). Survivorship (mean 6 SD) was calculated from cell numbers (B. saltans and C. campylum, both n 5 3) and from the number of active individuals (K. cochlearis, n 5 24). The figure of B. saltans was previously published in Matz et al. (2004). 2 d. Diets containing 10% CV017 reduced the abundance of K. cochlearis and B. calcyfloris significantly (by 44% and 74%, respectively) compared with the 0% CV017 treatment (Table 1). LT 50 values determined on a 25% CV017 diet Relationship between toxin sensitivity and biomassspecific clearance For an overall comparison of the toxicity effects on the different zooplankton taxa, which encompassed a size range spanning three orders of magnitude, taxa-specific toxin susceptibility (as measured by LT 50 ; Table 1) was related to the different specific bacterial consumption rates. Accordingly, the LT 50 values, determined with the 25% C. violaceum CV017 diet, were plotted as a function of biomass-specific clearance rate (Fig. 4). Generally, the concentration of toxin that an organism can tolerate depends on the toxicity of the prey item, the uptake rate, the assimilation efficiency, and the sensitivity to the toxin. Because violacein is primarily stored intracellularly by C. violaceum, the combined effect of grazer body weight and ingestion rate was tested.

6 1348 Deines et al. Table 1. Growth, feeding, and survival parameters of bacterivorous flagellates, ciliates, and rotifers on the violacein-producing bacterium C. violaceum CV017. LT 50 values (median lethal time) describe the time needed to kill 50% of the initial population. All values are given as means 6 standard deviation (n 5 3 for flagellates and ciliates, n 5 24 for rotifers). nd, not determined. * p, 0.05; ** p, 0.01; *** p, Growth rate (d 21 ) Maximum abundance (ml 21 ) LT 50 (h) Feeding selectivity Zooplankton species 0% CV017 10% CV017 0% CV017 10% CV017 25% CV017 50% CV017 Flagellates Bodo saltans *** * nd Ochromonas sp * ** Ciliates Colpidium campylum ** ** nd Tetrahymena pyriformis * * Rotifers Keratella cochlearis nd nd *** nd Brachionus calcyfloris nd nd *** nd Biomass-specific clearance rates were derived from the feeding rates measured in this study and from literature values on biovolumes, carbon contents (Pelegri et al. 1999), and feeding rates (nanoflagellates, Boenigk and Arndt 2000; ciliates, Curds and Cockburn 1971; Fenchel 1980; rotifers, Telesh et al. 1998; Nandini et al. 2003). The calculated log-log regression was significant (R , F 1, , p ) and revealed that higher concentrations of toxic bacteria are tolerated by filterfeeding zooplankton with high biomass-specific clearance rates that follow the power function LT BC 0.27, where BC is the biomass-specific clearance rate (nl ng 21 ). Discussion Rapid mortality of diverse zooplankton on violaceincontaining diets A bacterial diet containing the indole alkaloid violacein was found to be highly toxic for a broad spectrum of freshwater proto- and metazooplankton organisms. Food in which the ratio of violacein-producing C. violaceum was as low as 1 : 10 resulted in significant mortality rates across populations of nanoflagellates, ciliates, rotifers, and daphnids. Survivorship in the presence of toxic prey depends on the feeding selectivity and physiological sensitivity of the grazer as well as on the dose of ingested toxin. Our observation of decreasing survivorship despite the abundance of alternative nontoxic prey of high nutritional quality (e.g., 90% P. putida MM1), neutral selectivity coefficients, and decreasing cell numbers of VPB during grazing suggests high and unselective uptake rates of toxic bacteria. Despite indications of food discrimination in protists and rotifers (Kubanek et al. 2007; Montagnes et al. 2008), the zooplankton species tested were unable to detect the toxin before ingestion. The inability to discriminate against VPBs might be explained by the intracellular storage of violacein in the bacterial periplasm (between the inner and outer membranes; Matz et al. 2008). Possibly, periplasmic storage aids to increase the defense efficiency by minimizing autotoxicity and the loss of compounds into ambient water, but also by avoiding selection against violacein and ensuring direct contact with a potential consumer. The observed rapid decline in bacterivore survivorship on consumption of VPBs (Fig. 2) identifies violacein as a highly potent predator-targeting toxin, which is further supported by our calculations of a violacein content per bacterial cell in the low femtogram range. Microscopic analysis revealed that the uptake of a single violaceinproducing bacterium can cause the lysis of nanoflagellates and amoebae within,1 h (Matz et al. 2004, 2008). Recent findings suggest that the release of low amounts of violacein from ingested prey bacteria induces an apoptosis-like cell death mechanism in protozoan predators (Matz et al. 2008). Interestingly, violacein has been described to induce apoptosis in mammalian cell lines (Ferreira et al. 2004), which suggests a related mechanism underlying the violacein-induced decease of rotifers and daphnids. There is increasing evidence for the involvement of the antioxidant activity of violacein in the induction process (Konzen et al. 2006; C. Matz unpubl.). Whether such a mechanism is applicable to a wider scope of microbial pigments remains to be investigated. Taken together, the lack of consumer discrimination combined with the molecular induction of a eukaryotic cell death program appears to be responsible for the broad-spectrum and toxic activity of violacein-producing bacteria. Biomass-specific clearance determines sensitivity to violacein Besides the lethal effect on all zooplankton grazers tested, our experiments provided evidence of clear species-specific differences in bacterivore survivorship. For example, VPB-mediated growth suppression of the mixotrophic flagellate Ochromonas sp. was less pronounced than that of the strictly heterotrophic B. saltans. The lower toxin susceptibility of the ciliate T. pyriformis, as indicated by the high LT 50 values, seemed to be linked to markedly higher ingestion rates. High uptake rates and the continuous formation of food vacuoles apparently promote incomplete digestion and premature egestion of ingested bacteria in this species (Schlimme et al. 1997). This might not only increase bacterial survival but also minimize the digestive release of bacterial metabolites, including toxic ones such as violacein. Generally, the concentration of toxin that an organism can tolerate depends on the toxicity of the prey

7 Bioactive bacteria affect zooplankton 1349 Fig. 4. Allometric relationship between the biomass-specific clearance rate and toxin sensitivity of bacterivorous zooplankton (flagellates, ciliates, rotifers). Toxin sensitivity was calculated from LT 50 values (median lethal time after which the number of organisms is decreased by 50%). Least-squares regression line: log LT log BC Fig. 3. Survival of D. magna in the presence of violaceinproducing bacteria. (a) The diet of S. obliquus was supplemented with the violacein-producing C. violaceum CV017 and the violacein-deficient strain CV026. Nonfood controls were devoid of algal or bacterial food. Survivorship (mean 6 SD) of juvenile D. magna was calculated from the number of active individuals (n 5 15). (b) The dose effect curve of CV017 on D. magna juveniles and adults (both n 5 15). item, the uptake rate, the assimilation efficiency, and the sensitivity to the toxin. Because violacein is primarily stored intracellularly, the combined effect of grazer body weight and ingestion rate was tested. Similar to published plots on plankton susceptibility to dissolved cyanobacterial toxins as a function of body weight (Christoffersen 1996), we obtained an allometric relationship between biomassspecific clearance and bacterivore sensitivity to violacein (Fig. 4). Interestingly, the power function suggests that filter-feeding zooplankton with high biomass-specific clearance rates, such as rotifers and the ciliate T. pyriformis, tolerate higher concentrations of toxic bacteria in their diet. Apparently, the susceptibility of zooplankton grazers to violacein depends on both the efficiency at which the bacterium is ingested and the assimilation efficiency of the predator (cell or tissue). Violacein-producing bacteria in aquatic environments VPBs have been isolated from a range of aquatic habitats, which supports their widespread distribution. In addition, the repeated isolation of VPBs from grazing enrichment mesocosms might suggest that VPBs exhibit increased antipredator fitness in situ. Violacein is produced by at least six bacterial species of the genera Chromobacterium, Janthinobacterium, Pseudoalteromonas, and Microbulbifer (Matz et al. 2008). Although Pseudoalteromonas spp. are widely distributed in marine ecosystems (Skovhus et al. 2004), bacteria of the genera Janthinobacterium and Chromobacterium are frequently isolated from freshwater habitats (Curds and Vandyke 1966; Halda-Alija and Johnston 1999; Edwards et al. 2001). As for the vast majority of aquatic bacteria, current knowledge regarding the spatial and seasonal dynamics of VPBs in natural environments remains scarce. First, quantitative information comes from the members of the marine genus Pseudoalteromonas. The genus Pseudoalteromonas contains species with pronounced bioactivity, including many species with high antiprotozoal activities (Matz et al. 2008; C. Matz unpubl.) and three species that produce violacein. Population estimates of members of the genus Pseudoalteromonas have been made by means of real-time quantitative polymerase chain reaction (RTQ-PCR) targeting Pseudoalteromonas-specific 16S rrna genes (Skovhus et al. 2007). It was found from various samples collected off the Danish coast that Pseudoalteromonas spp. are abundant, especially in seawater plankton (3.4% abundance of Pseudoalteromonas 16S rrna genes of total eubacterial abundance). Recent bioassay studies screening for antiprotozoal activities in aquatic bacteria suggest that the production of predator-active compounds is not limited to VPB or Pseudoalteromonas spp. (Matz et al. 2008; C. Matz unpubl.). Hence, the relative bacterial abundances used in our study could seem realistic for natural plankton

8 1350 Deines et al. communities. Future RTQ-PCR studies targeting the biosynthetic gene cluster vioabcde are anticipated to elucidate the relative abundance and population dynamics of VPBs in natural systems. Total bacterial numbers as used in this study mimic those in meso- to eutrophic conditions (Wetzel 2001). All known violacein-producing bacterial taxa have been shown to produce violacein in a cell density dependent fashion and to benefit from increasing nutrient concentrations (Matz et al. 2008). The central role of population density for the biofilm-enhanced production of violacein is supported by the regulation of violacein biosynthesis in C. violaceum by acetylated homoserine lactones via the cvi quorum-sensing system (Blosser and Gray 2000). The cvi quorum-sensing system ensures that some phenotypic traits, including violacein biosynthesis, are expressed only when the bacterial population has reached a certain density. Although much needs to be discovered about the ecological niche occupied by VPBs in aquatic environments, cell density dependent gene regulation could illustrate the adaptive advantage of growth in clonal populations for the synthesis of antipredator compounds. As an analogy to phytoplankton blooms, localized blooms of VPBs (potentially in association with algal blooms) could suffice to reach the minimum quorum size. In biofilms, quorum-sensing systems have been demonstrated to be switched on in bacterial consortia of cells per aggregate (Dulla and Lindow 2008). Although not observed in our studies, isolates of C. violaceum have been described to produce copious amounts of exopolymers (Corpe 1953), which could favor an association with pelagic particulates and the formation of suspended clonal colonies. Analogies to microalgal zooplankton interactions On the basis of the increasing number of observations that some bloom-forming phytoplankton inhibit grazing via chemical defenses, it has been suggested that the failure of zooplankton to use phytoplankton biomass could contribute to the formation of algal blooms in some environments (Turner et al. 1998). One such example is diatoms, which have traditionally been considered an optimum food for zooplankton larval growth and the transfer of energy through the food chain to top carnivores. A series of recent studies indicate that some diatom species produce polyunsaturated aldehydes (PUA) that have a teratogenic effect on copepod offspring (Ianora et al. 2006). By interfering with reproductive success, PUAs appear to reduce grazing pressure of subsequent generations of zooplankters (Ianora et al. 2004). Interestingly, the suggested mode of action of copepod-active aldehydes is the induction of apoptosis through the activation of specific caspases that lead to the enzymatic breakdown of DNA (Romano et al. 2003). The fact that violacein is produced at cellular concentrations comparable to PUAs in diatoms and affects eukaryotic cells through a related molecular mechanism raises the question as to what extent VPBs and other bioactive bacteria might interfere with zooplankton offspring fitness in pelagic food webs. In freshwater, numerous investigations have demonstrated the strong effects of cyanobacterial metabolites like microcystins on aquatic organisms (Christoffersen 1996). It appears that sensitivity levels of proto- and metazooplankton to violacein, as determined in this study, fall in the same range as those detected for cyanobacterial microcystins, anatoxins, and nodularin. The high mortalities of D. magna when exposed to violacein concentrations as low as 9ngmL 21 suggest an even lower tolerance to violacein than to cyanobacterial toxins. As was the case in cyanobacteria daphnid studies (Lampert 1981; DeMott et al. 1991), our experiments showed that juvenile daphnids are more susceptible than adult animals to bioactive substances and that species-specific differences in sensitivity levels exist across zooplankters. Differential effects of toxic cyanobacteria on cladocerans and rotifers can reverse competitive outcomes among zooplankters and might change the species composition and size structure of zooplankton communities (Gilbert 1990; Kirk and Gilbert 1992). Moreover, phytoplankton metabolites appear to produce cascades throughout the food web by disrupting linkages between phytoplankton production and zooplankton growth (Christoffersen 1996), thus affecting not only community structure and species composition, but also fundamental pathways and rates of biogeochemical cycles. Similarly, bioactive bacteria could strongly influence the population dynamics of bacterivorous zooplankton. Comparable to inedible phytoplankton, blooms of predationresistant bacterial taxa that can temporarily dominate planktonic bacterial biomass can occur (Pernthaler et al. 2004). A reduction of zooplankton feeding activity because of the occurrence of toxic or inhibitory bacteria would directly undermine the grazing pressure on pico- and nanoplankton communities. Alternatively, the high sensitivity of HNFs to bacterial bioactives could lead to rapid changes in the abundance, size, and species distribution of nanoplankton communities, with secondary effects on higher trophic levels, because HNFs constitute an important food source for metazooplankton (Jürgens et al. 1996). Although much remains to be discovered about the production of bioactive bacterial metabolites in natural environments, our findings illustrate the importance of bacterial bioactives as fitness determinants for bacterivorous zooplankton and point out the need to elucidate their role in structuring aquatic food webs. We suggest that bacterial antipredator compounds are important for understanding microbial food webs, as well as the coupling of microbial and classical food webs with inherent biogeochemical processes. Acknowledgments We are grateful to two anonymous referees for their helpful comments. We thank V.C.L. Meyer for providing ciliate and rotifer cultures, L. 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